Culture medium additive for promoting growth of bacteroides microorganisms and application of culture medium additive

Abstract

The invention provides a culture medium additive for promoting growth of bacteroides microorganisms and application of the culture medium additive. The culture medium additive comprises an organic carbon source, an organic nitrogen source, an inorganic nitrogen source, magnesium salt, a regulator and a reducing agent. By adding the culture medium additive into a commercial culture medium, the rapid growth of bacteroides can be promoted, the viable count is increased, the culture period is shortened, a thought can be provided for the research on high-density fermentation of bacteroides in the future, and the research and development period is shortened.

Description

[0001] This application claims the benefit of priority to a prior application, application number 202410276179.7, filed with the State Intellectual Property Office of China on March 11, 2024, entitled “A culture medium additive for promoting the growth of Bacteroidetes microorganisms and its application.” The entire text of that prior application is incorporated herein by reference. Technical Field

[0002] The present invention relates to the technical field of microbial culture, in particular to a culture medium additive for promoting the growth of Bacteroidetes microorganisms and application thereof. Background Art

[0003] Intestinal microorganisms are closely related to host health. There is a rich flora in the human intestine. Although human intestinal microorganisms have been isolated and cultured on a large scale in recent years, 50% to 70% of intestinal microorganisms have not been isolated and cultured, or the existing culture medium is not suitable for increasing the biomass of certain difficult-to-culture intestinal bacterial genera.

[0004] Bacteroides is a non-spore-forming, Gram-negative, strictly anaerobic bacterium. It is an abundant genus in the human gut, comprising almost 25% of the human gut. One reason for its widespread presence is its ability to degrade a variety of host dietary and mucosal polysaccharides, as well as polysaccharides present on the surfaces of other gut microbes. Due to their potential role in promoting host health, some Bacteroides species have been considered candidates for next-generation probiotics (NGPs).

[0005] Parabacteroides is a core member of the human intestinal flora. It has the physiological characteristics of carbohydrate metabolism and secretion of short-chain fatty acids, and is closely related to human health and disease.

[0006] Alistipes is a genus of Gram-negative, obligately anaerobic bacteria in the class Bacteroidetes. It is a commensal in the intestine. It is straight or slightly curved, rod-shaped, 0.2–0.9 μm in diameter and 0.5–4 μm in length, with rounded ends. It does not form spores. Cells typically occur singly or in pairs, occasionally forming long filaments. Existing research suggests that Alistipes may have protective effects against certain diseases, including liver fibrosis, cancer immunotherapy, and cardiovascular disease.

[0007] In the isolation and culture of intestinal microorganisms, GAM, FAB, BHI, and RCM culture media are often used to screen anaerobic bacteria. The growth ability of Bacteroidetes in the four finished culture media varies, and a certain number of microorganisms can be cultured. Bacteroidetes can grow in the four finished culture media, but the growth is relatively slow and the number of viable bacteria is not high. According to literature reports, certain Bacteroidetes inhabiting the intestine can metabolize polysaccharides to provide nutrition for the host and other intestinal microorganisms. Appropriate nutrients are selected based on the metabolic characteristics of Bacteroidetes. Currently, research on the functional activity of Bacteroidetes has become a hot topic, but there are few studies on fermentation media for culturing Bacteroidetes. Although GAM, FAB, BHI, and RCM culture media can culture a certain amount of Bacteroidetes, a new culture medium is still needed to promote the rapid growth of strains. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the present invention provides a culture medium additive for promoting the growth of Bacteroidetes microorganisms and its application, which solves the problem that Bacteroidetes microorganisms grow slowly and have low viable counts in existing finished culture media.

[0009] In one aspect, the present invention provides a culture medium additive, which comprises: an organic carbon source, an organic nitrogen source, an inorganic nitrogen source, a magnesium salt, a regulator, and a reducing agent.

[0010] In one embodiment of the present invention, the organic carbon source comprises one or more of fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, cellobiose, N-acetyl-D-glucosamine, xylooligosaccharides, arabinose, mannose, and trehalose. Preferably, the organic carbon source comprises fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, cellobiose, and N-acetyl-D-glucosamine.

[0011] In one embodiment of the present invention, the organic nitrogen source comprises amino acids, and the amino acids comprise one or more of threonine, tryptophan, arginine and lysine. Preferably, the amino acids comprise threonine, tryptophan and arginine.

[0012] In one embodiment of the present invention, the inorganic nitrogen source includes ammonium salts and nitrates. Preferably, the ammonium salts include ammonium sulfate, ammonium acetate or ammonium chloride.

[0013] In one embodiment of the present invention, the magnesium salt includes magnesium nitrate and its hydrate, magnesium sulfate and its hydrate. Preferably, the magnesium salt includes magnesium sulfate heptahydrate.

[0014] In one embodiment of the present invention, the regulator includes phosphate, chloride, bicarbonate, and acetate. Preferably, the regulator includes one or more or all of sodium chloride, sodium bicarbonate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and sodium acetate.

[0015] In one embodiment of the present invention, the reducing agent comprises mercaptoethanol, ascorbic acid, glutathione, and cysteine ​​hydrochloride. Preferably, the reducing agent comprises cysteine ​​hydrochloride.

[0016] In the present invention, the organic carbon source is used to provide a carbon source for the growth of Bacteroidetes; the organic nitrogen source and the inorganic nitrogen source are used to provide a nitrogen source for the growth of Bacteroidetes; the magnesium salt is used to participate in the EMP (glycolysis pathway) and TCA (tricarboxylic acid cycle) pathways of Bacteroidetes; the balance regulator is used to maintain the potential difference and osmotic pressure of Bacteroidetes and adjust the pH, among which sodium acetate and the like also have the effect of selectively inhibiting miscellaneous bacteria; the reducing agent is used to remove free oxygen, which is beneficial to the growth and cultivation of anaerobic bacteria.

[0017] In one embodiment of the present invention, the culture medium additives include fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, cellobiose, N-acetyl-D-glucosamine, threonine, tryptophan, arginine, ammonium sulfate, magnesium sulfate heptahydrate, sodium chloride, sodium bicarbonate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium acetate, and cysteine ​​hydrochloride.

[0018] In one embodiment of the present invention, the culture medium additive is in the form of an aqueous solution, wherein the concentrations of the components are as follows: 2-30 g / L fructooligosaccharides, 3-40 g / L galacto-oligosaccharides, 5-50 g / L isomaltooligosaccharides, 5-40 g / L cellobiose, 12-80 g / L N-acetyl-D-glucosamine, 20-70 g / L threonine, 1-25 g / L tryptophan, 2-28 g / L arginine, 2-20 g / L ammonium sulfate, 0.1-2 g / L magnesium sulfate heptahydrate, 2-20 g / L sodium chloride, 10-40 g / L sodium bicarbonate, 10-35 g / L sodium acetate, 2-9 g / L potassium dihydrogen phosphate, 2-9 g / L dipotassium hydrogen phosphate, and 1-10 g / L cysteine ​​hydrochloride.

[0019] In one embodiment of the present invention, the concentrations of the components are as follows: 7.5-25 g / L fructooligosaccharides, 3.8-12 g / L galacto-oligosaccharides, 6.5-45 g / L isomaltooligosaccharides, 8-32 g / L cellobiose, 13.5-50 g / L N-acetyl-D-glucosamine, 25.5-55 g / L threonine, 2.6-15 g / L tryptophan, 3.8-18.8 g / L arginine, 2.8-17.8 g / L ammonium sulfate, 0.3-1.4 g / L magnesium sulfate heptahydrate, 4.5-13.5 g / L sodium chloride, 11.5-30 g / L sodium bicarbonate, 15.5-30 g / L sodium acetate, 2.5-5.5 g / L potassium dihydrogen phosphate, 2.5-5.5 g / L dipotassium hydrogen phosphate, and 1.5-7 g / L cysteine ​​hydrochloride.

[0020] In one embodiment of the present invention, the concentration of each component is as follows: 8-20 g / L fructooligosaccharides, 6.5-11 g / L galacto-oligosaccharides, 8.5-17.5 g / L isomaltooligosaccharides, 9.8-16.4 g / L cellobiose, 21-50 g / L N-acetyl-D-glucosamine, 27.5-52 g / L threonine, 3-10 g / L tryptophan, 4-12.8 g / L arginine, 4.5-13.5 g / L ammonium sulfate, 0.55-1.2 g / L magnesium sulfate heptahydrate, 6.5-9 g / L sodium chloride, 16.5-28 g / L sodium bicarbonate, 16.8-25.5 g / L sodium acetate, 3-5 g / L potassium dihydrogen phosphate, 3-5 g / L potassium hydrogen phosphate, and 2.5-6.3 g / L cysteine ​​hydrochloride.

[0021] In a second aspect, the present invention provides a method for preparing the above-mentioned culture medium additive, comprising weighing each component in the culture medium additive and dissolving each component in water.

[0022] The third aspect of the present invention provides a culture medium comprising a basal culture medium and the above-mentioned culture medium additive.

[0023] In one embodiment of the present invention, the basal culture medium is commercially available or prepared according to a public formula, including a solid culture medium, a liquid culture medium or a semi-solid culture medium.

[0024] In one embodiment of the present invention, the basal culture medium includes various commonly used and / or commercial basal culture media, including but not limited to GAM medium, FAB medium, BHI medium, and RCM medium.

[0025] In a fourth aspect, the present invention provides use of the above-mentioned culture medium additive and culture medium in culturing Bacteroidetes microorganisms, increasing the yield of Bacteroidetes bacteria and / or increasing the growth rate of Bacteroidetes bacteria.

[0026] In one embodiment of the present invention, the Bacteroidetes microorganisms include Bacteroides, Fusobacterium, Desulfovibrio, Ruminococcus, Ruminococcus, Oscillibacter, Alistipes, Parabacteroides, Anaerostipes, and Symbiobacterium.

[0027] In a specific embodiment of the present invention, the genus Bacteroides includes any one or more of Bacteroides xylanisolvens, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides caccae, and Bacteroides ovatus.

[0028] In a specific embodiment of the present invention, the Parabacteroides genus includes any one or more of Parabacteroides distasonis, Parabacteroides merdae, and Parabacteroides goldsteinii.

[0029] In a fifth aspect, the present invention provides a method for culturing Bacteroidetes microorganisms, comprising inoculating Bacteroidetes microorganisms into the above-mentioned culture medium for culturing.

[0030] In a sixth aspect, the present invention provides a method for increasing the bacterial yield and growth rate of Bacteroidetes microorganisms, comprising inoculating Bacteroidetes microorganisms into the above-mentioned culture medium for culturing.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] Adding the above-mentioned culture medium additive to commercial culture medium can promote the faster growth of Bacteroidetes microorganisms, increase the final biomass, shorten the culture cycle, and improve culture efficiency; while providing rich nutrients, it can further promote the growth of Bacteroidetes; it can also provide ideas for future high-density fermentation research of Bacteroidetes microorganisms and shorten the research and development cycle. The present invention improves the bacterial yield and growth rate of Bacteroidetes through the synergistic effect between the various components in the additive and between the various components of the additive and the basal culture medium. The additive of the present invention is suitable for all kinds of basal culture media and has universal applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1(a) shows the growth trend of Bacteroides xylanisolvens in different culture media; Figure 1(b) shows the autofluorescence units of Bacteroides xylanisolvens after 24 hours of culture in different culture media.

[0034] Figure 2(a) shows the growth trend of Bacteroides fragilis in different culture media; Figure 2(b) shows the autofluorescence units of Bacteroides fragilis after 24 hours of culture in different culture media.

[0035] Figure 3(a) shows the growth trend of Bacteroides thetaiotaomicron in different culture media; Figure 3(b) shows the autofluorescence units of Bacteroides thetaiotaomicron after 24 hours of culture in different culture media.

[0036] Figure 4(a) shows the growth trend of Bacteroides caccae in different culture media; Figure 4(b) shows the autofluorescence units of Bacteroides caccae after 24 hours of culture in different culture media.

[0037] Figure 5(a) shows the growth trend of Bacteroides ovatus in different culture media; Figure 5(b) shows the autofluorescence units of Bacteroides ovatus after 24 hours of culture in different culture media.

[0038] Figure 6(a) shows the growth trend of Parabacteroides distasonis in different culture media; Figure 6(b) shows the autofluorescence units of Parabacteroides distasonis after 24 hours of culture in different culture media.

[0039] Figure 7(a) shows the growth trend of Parabacteroides merdae in different culture media; Figure 7(b) shows the autofluorescence units of Parabacteroides merdae after 24 hours of culture in different culture media.

[0040] Figure 8(a) shows the growth trend of Parabacteroides goldsteinii in different culture media; Figure 8(b) shows the autofluorescence units of Parabacteroides goldsteinii after 24 hours of culture in different culture media DETAILED DESCRIPTION

[0041] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0042] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0043] Technical terms:

[0044] The autofluorescence unit (AFU) is a new microbial counting unit introduced by a rapid counting method. Instead of waiting for sufficient growth to detect colony-forming units (CFU), the fluorescence counting system uses scattered light to count particles, classifying the detected particles as either bioluminescent or non-bioluminescent.

[0045] The bacterial strains and commercial culture medium used in the present invention are as follows:

[0046] The Bacteroides and Parabacteroides used were screened and isolated from intestinal microbial samples of healthy people.

[0047] Improved GAM broth medium was purchased from Haibo; brain heart infusion broth (BHI) medium was purchased from OXOID; caustic anaerobic broth (FAB) medium was purchased from NEOGEN; and reinforced Clostridium difficile (RCM) medium was purchased from OXOID.

[0048] Specific ingredients of modified GAM broth medium:

[0049]

[0050] BHI medium specific components:

[0051]

[0052]

[0053] Specific ingredients of caustic anaerobic broth (FAB) medium:

[0054] FAB medium components Unit (g / L) Mixed peptone 15 Sodium chloride 2.5 L-Cysteine ​​Hydrochloride 0.5 Sodium bicarbonate 0.4 Sodium thioglycolate 0.5 Yeast extract powder 10 Hemin 0.005 Resazurin 0.001 Vitamin K 0.0005 agar 0.75

[0055] Reinforced Clostridium difficile (RCM) medium specific ingredients:

[0056] RCM medium components Unit (g / L) yeast powder 13 Peptone 10 Soluble starch 1 glucose 5 L-Cysteine ​​Hydrochloride 0.5 Sodium chloride 5 Sodium acetate 3 agar 0.5

[0057] Example 1 Preparation of PS solution (100 mL)

[0058] PS solution formula 1: 1g fructooligosaccharide, 1g galacto-oligosaccharide, 1g isomaltooligosaccharide, 1g cellobiose, 5g N-acetyl-D-glucosamine, 5g threonine, 0.5g tryptophan, 0.5g arginine, 0.9g ammonium sulfate, 0.09g magnesium sulfate heptahydrate, 0.9g sodium chloride, 3g sodium bicarbonate, 2.46g sodium acetate, 0.45g potassium dihydrogen phosphate, 0.45g dipotassium hydrogen phosphate, 0.5g L-cysteine ​​hydrochloride

[0059] PS solution formula 2: 1.45g fructooligosaccharides, 0.65g galacto-oligosaccharides, 0.8g isomaltooligosaccharides, 1.25g cellobiose, 1.35g N-acetyl-D-glucosamine, 3.5g threonine, 0.26g tryptophan, 0.38g arginine, 0.28g ammonium sulfate, 0.03g magnesium sulfate heptahydrate, 0.45g sodium chloride, 1.25g sodium bicarbonate, 1.55g sodium acetate, 0.25g potassium dihydrogen phosphate, 0.25g dipotassium hydrogen phosphate, 0.15g L-cysteine ​​hydrochloride

[0060] PS solution formula 3: 0.75g fructooligosaccharides, 1.2g galacto-oligosaccharides, 0.65g isomaltooligosaccharides, 1.5g cellobiose, 4.25g N-acetyl-D-glucosamine, 7g threonine, 0.56g tryptophan, 1.88g arginine, 1.05g ammonium sulfate, 0.12g magnesium sulfate heptahydrate, 0.75g sodium chloride, 4g sodium bicarbonate, 1.55g sodium acetate, 0.38g potassium dihydrogen phosphate, 0.38g dipotassium hydrogen phosphate, 0.18g L-cysteine ​​hydrochloride

[0061] PS solution formula 4: 2.5g fructooligosaccharides, 0.78g galacto-oligosaccharides, 3.25g isomaltooligosaccharides, 0.8g cellobiose, 4.6g N-acetyl-D-glucosamine, 2.55g threonine, 1.2g tryptophan, 0.62g arginine, 0.34g ammonium sulfate, 0.08g magnesium sulfate heptahydrate, 1.25g sodium chloride, 2.85g sodium bicarbonate, 3g sodium acetate, 0.55g potassium dihydrogen phosphate, 0.55g dipotassium hydrogen phosphate, 0.3g L-cysteine ​​hydrochloride

[0062] PS solution formula 5: 1.15g fructooligosaccharides, 0.38g galacto-oligosaccharides, 4.5g isomaltooligosaccharides, 3.2g cellobiose, 2.5g N-acetyl-D-glucosamine, 5.5g threonine, 0.65g tryptophan, 0.82g arginine, 1.78g ammonium sulfate, 0.08g magnesium sulfate heptahydrate, 0.45g sodium chloride, 1.85g sodium bicarbonate, 1.55g sodium acetate, 0.25g potassium dihydrogen phosphate, 0.25g dipotassium hydrogen phosphate, 0.15g L-cysteine ​​hydrochloride

[0063] PS solution formula 6: 0.2g fructooligosaccharides, 4g galacto-oligosaccharides, 0.5g isomaltooligosaccharides, 4g cellobiose, 8g N-acetyl-D-glucosamine, 2g threonine, 0.1g tryptophan, 0.2g arginine, 0.2g ammonium sulfate, 0.01g magnesium sulfate heptahydrate, 0.2g sodium chloride, 1g sodium bicarbonate, 3.5g sodium acetate, 0.2g potassium dihydrogen phosphate, 0.2g dipotassium hydrogen phosphate, 0.1g L-cysteine ​​hydrochloride

[0064] PS solution formula 7: 2.6g fructooligosaccharides, 3.5g galacto-oligosaccharides, 3.8g isomaltooligosaccharides, 0.5g cellobiose, 1.2g N-acetyl-D-glucosamine, 2g threonine, 2.5g tryptophan, 2.8g arginine, 1.2g ammonium sulfate, 0.2g magnesium sulfate heptahydrate, 2g sodium chloride, 4g sodium bicarbonate, 1.5g sodium acetate, 0.9g potassium dihydrogen phosphate, 0.9g dipotassium hydrogen phosphate, 1g L-cysteine ​​hydrochloride

[0065] PS solution formula 8: 1.85g fructooligosaccharides, 3.5g galacto-oligosaccharides, 5g isomaltooligosaccharides, 0.5g cellobiose, 1.2g N-acetyl-D-glucosamine, 3g threonine, 2.5g tryptophan, 2.4g arginine, 1.5g ammonium sulfate, 0.15g magnesium sulfate heptahydrate, 1.7g sodium chloride, 4g sodium bicarbonate, 1.9g sodium acetate, 0.8g potassium dihydrogen phosphate, 0.8g dipotassium hydrogen phosphate, 1g L-cysteine ​​hydrochloride

[0066] PS solution formula 9: 3g fructooligosaccharides, 2g galacto-oligosaccharides, 4.5g isomaltooligosaccharides, 0.8g cellobiose, 1.2g N-acetyl-D-glucosamine, 2g threonine, 2.2g tryptophan, 1.5g arginine, 2g ammonium sulfate, 0.2g magnesium sulfate heptahydrate, 1.5g sodium chloride, 4g sodium bicarbonate, 1g sodium acetate, 0.9g potassium dihydrogen phosphate, 0.9g dipotassium hydrogen phosphate, 1g L-cysteine ​​hydrochloride

[0067] Example 2 Growth of Bacteroides xylanisolvens in different culture media

[0068] According to the above PS solution formula 1, weigh the reagents, dissolve them with pure water, heat them in a microwave oven to fully dissolve them and make the volume to 100 mL. Finally, filter and sterilize to obtain the PS solution for use.

[0069] 10 mL of PS solution was respectively pipetted into 90 mL of commercial liquid culture medium GAM, BHI, FAB, and RCM sterilized by high-pressure steam to obtain GAM+PS, BHI+PS, FAB+PS, and RCM+PS liquid culture media.

[0070] A strain of Bacteroides xylanisolvens (screened, isolated and cultured from fecal samples of healthy people) was taken out from an ultra-low temperature freezer at -80°C, and the strain was streaked on a FAB+PS solid plate. The plate was cultured for about 36 hours until a single colony grew. The single colony was picked and transferred to FAB+PS liquid, cultured for 24 hours, and passed for 2-3 generations. The resulting bacterial liquid was used as a seed liquid for later use. Bacteroides xylanisolvens was inoculated into 8 liquid culture media including GAM, BHI, FAB, RCM, GAM+PS, BHI+PS, FAB+PS, and RCM+PS at a 5% inoculation rate, and transferred to a honeycomb well plate and sealed according to the corresponding experimental number. All the above operations were performed in an anaerobic workstation at 37°C; the honeycomb plate was placed in an automatic growth curve analyzer to monitor the OD regularly. 600 The culture temperature was set to 37°C for 24 h. The bacterial suspensions cultured in different media for 24 h were retained and the autofluorescence units were measured using a flow cytometer.

[0071] The results are shown in FIG1( a ), where the OD values ​​of Bacteroides xylanisolvens in 4 kinds of GAM+PS, BHI+PS, FAB+PS, and RCM+PS liquid culture media supplemented with 10% PS solution of the present invention for 24 h were 0. 600 The values ​​were higher than those in the basal medium, and the growth conditions were effectively improved. Figure 1(b) shows that the autofluorescence units of GAM+PS and FAB+PS after 24 h of culture were higher, both greater than 9×10 9 AFU / mL.

[0072] Example 3 Growth of Bacteroides fragilis in different culture media

[0073] According to the above PS solution formula 2, the reagents were weighed, dissolved with pure water, heated in a microwave oven to fully dissolve and the volume was adjusted to 100 mL. Finally, the PS solution was filtered and sterilized to obtain a standby PS solution.

[0074] 10 mL of PS solution was respectively pipetted into 90 mL of commercial liquid culture medium GAM, BHI, FAB, and RCM sterilized by high-pressure steam to obtain GAM+PS, BHI+PS, FAB+PS, and RCM+PS liquid culture media.

[0075] A strain of Bacteroides fragilis (screened, isolated and cultured from fecal samples of healthy people) was taken out from an ultra-low temperature freezer at -80°C, and the strain was streaked onto a FAB+PS solid plate and cultured for about 36 hours until a single colony grew. The single colony was picked and transferred to FAB+PS liquid, cultured for 24 hours, and passed 2-3 generations. The obtained bacterial liquid was used as a seed liquid for standby use. Bacteroides fragilis was inoculated into 8 liquid culture media including GAM, BHI, FAB, RCM, GAM+PS, BHI+PS, FAB+PS, and RCM+PS at a 5% inoculation rate, and transferred to a honeycomb well plate and sealed according to the corresponding experimental number. All the above operations were carried out in a 37°C anaerobic workstation; the honeycomb plate was placed in an automatic growth curve analyzer to monitor the OD regularly. 600 The culture temperature was set to 37°C for 24 h. The bacterial suspensions cultured in different media for 24 h were retained and the autofluorescence units were measured using a flow cytometer.

[0076] The results show that in Figure 2(a), Bacteroides fragilis grew fastest in the FAB+PS liquid medium supplemented with 10% PS solution of the present invention, and the growth in GAM+PS, BHI+PS, and RCM+PS was improved to varying degrees compared with the original basal medium. In Figure 2(b), the 24h autofluorescence unit was the highest in FAB+PS, reaching 9.78×10 9 AFU / mL.

[0077] Example 4 Growth of Bacteroides thetaiotaomicron in different culture media

[0078] According to the above PS solution formula 3, the reagents were weighed, dissolved in pure water, heated in a microwave oven to fully dissolve and the volume was adjusted to 100 mL. Finally, the PS solution was filtered and sterilized to obtain a standby PS solution.

[0079] 10 mL of PS solution was respectively pipetted into 90 mL of commercial liquid culture medium GAM, BHI, FAB, and RCM sterilized by high-pressure steam to obtain GAM+PS, BHI+PS, FAB+PS, and RCM+PS liquid culture media.

[0080] A strain of Bacteroides thetaiotaomicron (screened, isolated and cultured from fecal samples of healthy people) was taken out from an ultra-low temperature freezer at -80°C. The strain was streaked onto a FAB+PS solid plate and cultured for about 36 hours until a single colony grew. The single colony was picked and transferred to a FAB+PS liquid, cultured for 24 hours, and passed for 2-3 generations. The resulting bacterial liquid was used as a seed liquid for later use. Bacteroides thetaiotaomicron was inoculated into 8 liquid culture media, including GAM, BHI, FAB, RCM, GAM+PS, BHI+PS, FAB+PS, and RCM+PS, at a 5% inoculation rate. The plates were transferred to honeycomb well plates and sealed according to the corresponding experimental numbers. All the above operations were performed in an anaerobic workstation at 37°C. The honeycomb plates were placed in an automatic growth curve analyzer to monitor the OD at regular intervals. 600 The culture temperature was set to 37°C for 24 h. The bacterial suspensions cultured in different media for 24 h were retained and the autofluorescence units were measured using a flow cytometer.

[0081] The results are shown in Figure 3(a). The growth of Bacteroides thetaiotaomicron in GAM+PS, BHI+PS, and FAB+PS was optimized. Among them, the growth rate in FAB+PS was the fastest and the OD 600 The highest; Figure 3 (b) The 24h autofluorescence units of GAM+PS and FAB+PS were both higher than 7×10 9 AFU / mL.

[0082] Example 5 Growth of Bacteroides caccae in different culture media

[0083] According to the above PS solution formula 4, the reagents were weighed, dissolved with pure water, heated in a microwave oven to fully dissolve and the volume was adjusted to 100 mL. Finally, the PS solution was filtered and sterilized to obtain a standby PS solution.

[0084] 10 mL of PS solution was respectively pipetted into 90 mL of commercial liquid culture medium GAM, BHI, FAB, and RCM sterilized by high-pressure steam to obtain GAM+PS, BHI+PS, FAB+PS, and RCM+PS liquid culture media.

[0085] A strain of Bacteroides caccae (screened, isolated and cultured from fecal samples of healthy people) was taken out from an ultra-low temperature freezer at -80°C, and the strain was streaked on a FAB+PS solid plate and cultured for about 36 hours until a single colony grew. The single colony was picked and transferred to a FAB+PS liquid, cultured for 24 hours, and passed 2-3 generations. The resulting bacterial liquid was used as a seed liquid for standby use. Bacteroides caccae was inoculated into 8 liquid culture media including GAM, BHI, FAB, RCM, GAM+PS, BHI+PS, FAB+PS, and RCM+PS at a 5% inoculation rate, and transferred to a honeycomb well plate and sealed according to the corresponding experimental number. All the above operations were performed in an anaerobic workstation at 37°C; the honeycomb plate was placed in an automatic growth curve analyzer to monitor the OD regularly. 600 The culture temperature was set to 37°C for 24 h. The bacterial suspensions cultured in different media for 24 h were retained and the autofluorescence units were measured using a flow cytometer.

[0086] The results are shown in Figure 4(a). The growth conditions of Bacteroides caccae in GAM+PS, BHI+PS, FAB+PS, and RCM+PS culture media supplemented with the PS solution of the present invention were improved compared with the four basic culture media of GAM, BHI, FAB, and RCM. Figure 4(b) shows that the maximum value of the autofluorescence unit was reached fastest in the culture medium FAB+PS.

[0087] Example 6 Growth of Bacteroides ovatus in different culture media

[0088] According to the above PS solution formula 5, the reagents were weighed, dissolved in pure water, heated in a microwave oven to fully dissolve and the volume was adjusted to 100 mL. Finally, the PS solution was filtered and sterilized to obtain a standby PS solution.

[0089] 10 mL of PS solution was respectively pipetted into 90 mL of commercial liquid culture medium GAM, BHI, FAB, and RCM sterilized by high-pressure steam to obtain GAM+PS, BHI+PS, FAB+PS, and RCM+PS liquid culture media.

[0090] A strain of Bacteroides ovatus (screened, isolated and cultured from fecal samples of healthy people) was taken out from an ultra-low temperature freezer at -80°C, and the strain was streaked onto a FAB+PS solid plate and cultured for about 36 hours until a single colony grew. The single colony was picked and transferred to FAB+PS liquid, cultured for 24 hours, and passed 2-3 generations. The resulting bacterial liquid was used as a seed liquid for standby use. Bacteroides ovatus was inoculated into 8 liquid culture media including GAM, BHI, FAB, RCM, GAM+PS, BHI+PS, FAB+PS, and RCM+PS at a 5% inoculation rate, and transferred to a honeycomb plate and sealed according to the corresponding experimental number. All the above operations were performed in a 37°C anaerobic workstation; the honeycomb plate was placed in an automatic growth curve analyzer to monitor OD regularly. 600 The culture temperature was set to 37°C for 24 h. The bacterial suspensions cultured in different media for 24 h were retained and the autofluorescence units were measured using a flow cytometer.

[0091] The results are shown in Figure 5(a). The growth conditions of Bacteroides ovatus in the basal medium with the additive PS solution of the present invention, GAM+PS, BHI+PS, and FAB+PS, were improved compared with those in the basal medium with GAM, BHI, and FAB. The maximum OD was reached in the FAB+PS medium. 600 The time taken to obtain the value is the shortest; Figure 5(b) shows that the 24h autofluorescence unit of FAB+PS is the highest, reaching 9.48×10 9 AFU / mL.

[0092] Example 7 Growth of Parabacteroides distasonis in different culture media

[0093] According to the above PS solution formula 6, weigh the reagents, dissolve them with pure water, heat them in a microwave oven to fully dissolve them and make the volume to 100 mL, and finally filter and sterilize to obtain the PS solution for use.

[0094] 10 mL of PS solution was respectively pipetted into 90 mL of commercial liquid culture medium GAM, BHI, FAB, and RCM sterilized by high-pressure steam to obtain GAM+PS, BHI+PS, FAB+PS, and RCM+PS liquid culture media.

[0095] A strain of Parabacteroides distasonis (screened, isolated and cultured from fecal samples of healthy people) was taken out from an ultra-low temperature freezer at -80°C, and the strain was streaked onto a FAB+PS solid plate and cultured for about 36 hours until a single colony grew. The single colony was picked and transferred to a FAB+PS liquid, cultured for 24 hours, and passed for 2-3 generations. The resulting bacterial liquid was used as a seed liquid for standby use. Parabacteroides distasonis was inoculated into 8 liquid culture media, GAM, BHI, FAB, RCM, GAM+PS, BHI+PS, FAB+PS, and RCM+PS, at a 5% inoculation rate, and transferred to a honeycomb well plate and sealed according to the corresponding experimental number. All the above operations were performed in an anaerobic workstation at 37°C; the honeycomb plate was placed in an automatic growth curve analyzer to monitor the OD regularly. 600 The culture temperature was set to 37°C for 24 h. The bacterial suspensions cultured in different media for 24 h were retained and the autofluorescence units were measured using a flow cytometer.

[0096] As shown in Figure 6(a), the growth of Parabacteroides distasonis in the four basal culture media after adding PS solution, including GAM+PS, BHI+PS, FAB+PS, and RCM+PS, was effectively improved. In FAB+PS, the growth reached the stable phase in the shortest time and the OD 600 Maintained at the highest level; Figure 6(b) shows that the autofluorescence unit at 24h in FAB+PS was the highest, followed by BHI+PS.

[0097] Example 8 Growth of Parabacteroides merdae in different culture media

[0098] According to the above PS solution formula 7, the reagents were weighed, dissolved in pure water, heated in a microwave oven to fully dissolve and the volume was adjusted to 100 mL. Finally, the PS solution was filtered and sterilized to obtain a standby PS solution.

[0099] 10 mL of PS solution was respectively pipetted into 90 mL of commercial liquid culture medium GAM, BHI, FAB, and RCM sterilized by high-pressure steam to obtain GAM+PS, BHI+PS, FAB+PS, and RCM+PS liquid culture media.

[0100] A strain of Parabacteroides merdae (screened, isolated and cultured from fecal samples of healthy people) was taken out from an ultra-low temperature freezer at -80°C, and the strain was streaked onto a FAB+PS solid plate and cultured for about 36 hours until a single colony grew. The single colony was picked and transferred to a FAB+PS liquid, cultured for 24 hours, and passed for 2-3 generations. The resulting bacterial liquid was used as a seed liquid for standby use. Parabacteroides merdae was inoculated into 8 liquid culture media, GAM, BHI, FAB, RCM, GAM+PS, BHI+PS, FAB+PS, and RCM+PS, at a 5% inoculation rate, and transferred to a honeycomb well plate and sealed according to the corresponding experimental number. All the above operations were performed in an anaerobic workstation at 37°C; the honeycomb plate was placed in an automatic growth curve analyzer to monitor the OD regularly. 600 The culture temperature was set to 37°C for 24 h. The bacterial suspensions cultured in different media for 24 h were retained and the autofluorescence units were measured using a flow cytometer.

[0101] As shown in Figure 7(a), the growth trend of Parabacteroides merdae in FAB+PS medium was better than that in the other seven media, and it reached the stable growth phase in the shortest time. The growth in BHI+PS, GAM+PS, and RCM+PS media was relatively slow. In Figure 7(b), the 24-hour autofluorescence units of GAM+PS, BHI+PS, FAB+PS, and RCM+PS were all higher than those of their respective basal media. In addition, the 24-hour autofluorescence unit of FAB+PS reached the highest level of 3.28×10 9 AFU / mL.

[0102] Example 9 Growth of Parabacteroides goldsteinii in different culture media

[0103] According to the above PS solution formula 8, the reagents were weighed, dissolved in pure water, heated in a microwave oven to fully dissolve and the volume was adjusted to 100 mL. Finally, the PS solution was filtered and sterilized to obtain a standby PS solution.

[0104] 10 mL of PS solution was respectively pipetted into 90 mL of commercial liquid culture medium GAM, BHI, FAB, and RCM sterilized by high-pressure steam to obtain GAM+PS, BHI+PS, FAB+PS, and RCM+PS liquid culture media.

[0105] A strain of Parabacteroides goldsteinii (screened, isolated and cultured from fecal samples of healthy people) was taken out from an ultra-low temperature freezer at -80°C, and the strain was streaked onto a FAB+PS solid plate and cultured for about 36 hours until a single colony grew. The single colony was picked and transferred to FAB+PS liquid, cultured for 24 hours, and passed 2-3 generations. The resulting bacterial liquid was used as a seed liquid for standby use. Parabacteroides goldsteinii was inoculated into 8 liquid culture media, GAM, BHI, FAB, RCM, GAM+PS, BHI+PS, FAB+PS, and RCM+PS, at a 5% inoculation rate, and transferred to a honeycomb plate and sealed according to the corresponding experimental number. All the above operations were performed in an anaerobic workstation at 37°C; the honeycomb plate was placed in an automatic growth curve analyzer to monitor the OD regularly. 600 The culture temperature was set to 37°C for 24 h. The bacterial suspensions cultured in different media for 24 h were retained and the autofluorescence units were measured using a flow cytometer.

[0106] As shown in Figure 8(a), the growth of Parabacteroides goldsteinii in FAB+PS and BHI+PS was significantly better than that in the basal medium. In FAB+PS, the stable growth phase was reached after 8 h, and the OD 600 The value reaches above 1.4; Figure 8(b)

[0107] The 24h autofluorescence units of GAM, BHI, FAB, and RCM were close to the same level, and the 24h autofluorescence unit of FAB+PS was the highest, reaching 2.28×10 9 AFU / mL.

[0108] Example 10 Growth of Alistipes onderdonkii in different culture media

[0109] According to the above PS solution formula 9, the reagents were weighed, dissolved in pure water, heated in a microwave oven to fully dissolve and the volume was adjusted to 100 mL. Finally, the PS solution was filtered and sterilized to obtain a standby PS solution.

[0110] 10 mL of PS solution was pipetted into 90 mL of commercial liquid culture medium FAB sterilized by high-pressure steam to obtain FAB+PS liquid culture medium.

[0111] Take out a strain of Alistipes onderdonkii (screened, isolated and cultured from fecal samples of healthy people) from a -80°C ultra-low temperature freezer, streak the strain on a FAB+PS solid plate, culture for about 36 hours until a single colony grows, pick a single colony and transfer it to FAB+PS liquid, culture for 24 hours, pass 2-3 generations, and use the resulting bacterial liquid as a seed liquid for later use. Inoculate Alistipes onderdonkii into FAB and FAB+PS liquid culture media at a 5% inoculum volume, culture at 37°C for 24 hours. The above operations were performed in an anaerobic workstation, and samples were taken after 24 hours to measure the OD 600 value.

[0112] Alistipes onderdonkii only grew in FAB+PS 600 At around 0.33, there was almost no growth in FAB medium. The results are shown in Table 1.

[0113] Table 1 Growth of Alistipes onderdonkii strains in two culture media

[0114]

[0115]

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. 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 purpose 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 additive, characterized in that The culture medium additives include: an organic carbon source, an organic nitrogen source, an inorganic nitrogen source, a magnesium salt, a regulator, and a reducing agent.

2. The culture medium additive according to claim 1, wherein The organic carbon source includes one or more of fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, cellobiose, N-acetyl-D-glucosamine, xylooligosaccharides, arabinose, mannose, and trehalose; preferably, the organic carbon source includes fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, cellobiose, and N-acetyl-D-glucosamine; The organic nitrogen source includes one or more of threonine, tryptophan, arginine and lysine; preferably includes threonine, tryptophan and arginine; The inorganic nitrogen source includes ammonium salts and nitrates. Preferably, the ammonium salts include ammonium sulfate, ammonium acetate or ammonium chloride. The magnesium salt includes magnesium nitrate and its hydrate, magnesium sulfate and its hydrate; preferably, the magnesium salt includes magnesium sulfate heptahydrate; The regulator includes phosphate, chloride, bicarbonate, acetate, preferably, the regulator includes one or more or all of sodium chloride, sodium bicarbonate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and sodium acetate; The reducing agent includes mercaptoethanol, ascorbic acid, glutathione, and cysteine ​​hydrochloride; preferably, the reducing agent includes cysteine ​​hydrochloride.

3. The culture medium additive according to claim 2, wherein The culture medium additives include fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, cellobiose, N-acetyl-D-glucosamine, threonine, tryptophan, arginine, ammonium sulfate, magnesium sulfate heptahydrate, sodium chloride, sodium bicarbonate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium acetate, and cysteine ​​hydrochloride.

4. The culture medium additive according to claim 3, wherein The culture medium additive is in the form of an aqueous solution, wherein the concentrations of the components are as follows: 2-30 g / L fructooligosaccharides, 3-40 g / L galacto-oligosaccharides, 5-50 g / L isomaltooligosaccharides, 5-40 g / L cellobiose, 12-80 g / L N-acetyl-D-glucosamine, 20-70 g / L threonine, 1-25 g / L tryptophan, 2-28 g / L arginine, 2-20 g / L ammonium sulfate, 0.1-2 g / L magnesium sulfate heptahydrate, 2-20 g / L sodium chloride, 10-40 g / L sodium bicarbonate, 10-35 g / L sodium acetate, 2-9 g / L potassium dihydrogen phosphate, 2-9 g / L dipotassium hydrogen phosphate, and 1-10 g / L cysteine ​​hydrochloride; Preferably, the concentration of each component is as follows: 7.5-25 g / L fructooligosaccharides, 3.8-12 g / L galacto-oligosaccharides, 6.5-45 g / L isomaltooligosaccharides, 8-32 g / L cellobiose, 13.5-50 g / L N-acetyl-D-glucosamine, 25.5-55 g / L threonine, 2.6-6.5 g / L tryptophan, 3.8-18.8 g / L arginine, 2.8-17.8 g / L ammonium sulfate, 0.3-1.2 g / L magnesium sulfate heptahydrate, 4.5-9.5 g / L sodium chloride, 11.5-30 g / L sodium bicarbonate, 15.5-30 g / L sodium acetate, 2.5-5.5 g / L potassium dihydrogen phosphate, 2.5-5.5 g / L dipotassium hydrogen phosphate, and 1.5-5 g / L cysteine ​​hydrochloride. Preferably, the concentration of each component is as follows: 8-20 g / L fructooligosaccharides, 6.5-11 g / L galacto-oligosaccharides, 8.5-17.5 g / L isomaltooligosaccharides, 9.8-16.4 g / L cellobiose, 21-50 g / L N-acetyl-D-glucosamine, 27.5-52 g / L threonine, 3-10 g / L tryptophan, 4-12.8 g / L arginine, 4.5-13.5 g / L ammonium sulfate, 0.55-1.2 g / L magnesium sulfate heptahydrate, 6.5-9 g / L sodium chloride, 16.5-28 g / L sodium bicarbonate, 16.8-25.5 g / L sodium acetate, 3-5 g / L potassium dihydrogen phosphate, 3-5 g / L dipotassium hydrogen phosphate, and 2.5-6.3 g / L cysteine ​​hydrochloride.

5. The method for preparing the culture medium additive according to any one of claims 1 to 4, characterized in that: The method comprises weighing each component in the culture medium additive and dissolving each component in water.

6. A culture medium, characterized in that The method comprises a basal culture medium and the culture medium additive according to any one of claims 1 to 4.

7. The culture medium according to claim 6, wherein The basal culture medium includes any one or more of GAM culture medium, FAB culture medium, BHI culture medium, and RCM culture medium.

8. Use of the culture medium additive according to any one of claims 1 to 4 or the culture medium according to any one of claims 6 to 7 in culturing Bacteroidetes microorganisms, increasing the yield of Bacteroidetes bacteria and / or increasing the growth rate of Bacteroidetes bacteria; Preferably, the Bacteroidetes microorganisms include Bacteroides, Fusobacterium, Desulfovibrio, Ruminococcus, Ruminococcus, Oscillibacter, Alistipes, Parabacteroides, Anaerostipes, and Symbiobacterium; Preferably, the genus Bacteroides includes any one or more of Bacteroides xylanisolvens, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides caccae, and Bacteroides ovatus; Preferably, the Parabacteroides genus includes any one or more of Parabacteroides distasonis, Parabacteroidesmerdae, and Parabacteroides goldsteinii.

9. A method for culturing Bacteroidetes microorganisms, characterized in that: The method comprises inoculating Bacteroides microorganisms and culturing the microorganisms in the culture medium according to any one of claims 6 to 7.

10. A method for increasing the bacterial yield and growth rate of Bacteroidetes microorganisms, characterized in that: The method comprises inoculating Bacteroides microorganisms and culturing the microorganisms in the culture medium according to any one of claims 6 to 7.

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