Fermentation medium and fermentation process of Blautia

Abstract

The present invention relates to the field of microbial culture. Specifically, the present invention relates to a high-density fermentation medium for Blautia and a fermentation method using the fermentation medium. The fermentation medium and the fermentation method of the present invention can significantly increase the bacterial mass of Blautia, thereby achieving low-cost and high-efficiency production of Blautia.

Description

Technical Field

[0001] The present invention relates to the field of microbial culture, and in particular to a non-animal-derived culture medium suitable for Blautia, especially Blautia elongata, and a fermentation process using the culture medium. Background Art

[0002] Blautia producta belongs to the genus Blautia and is a strictly anaerobic bacterium. Blautia producta is usually round or oval in shape, grows at a temperature range of 20°C-40°C, and can grow in a pH range of 6-8. At the same time, Blautia producta has heterotrophic and autotrophic characteristics and can use CO or H2 / CO2 as an energy source.

[0003] Since its discovery, Blautia has attracted widespread attention from researchers for its effectiveness in alleviating inflammation and metabolic diseases such as obesity and diabetes. Studies have found that the abundance of Blautia in the cecal mucosal flora of patients with inflammatory bowel disease (IBD) and Crohn's disease (CD) is significantly lower than that of healthy people. K. Lippert et al. found that the abundance of Blautia is correlated with serum glucose and insulin levels, which means that it plays a role in host glucose metabolism. In addition, some researchers have found that Blautia plays a role in insulin resistance in obese children.

[0004] In recent years, the biotransformation and metabolism of herbal plants and functional foods by Blautia have attracted the attention of researchers. Exploring the biotransformation of Blautia is crucial for the development of new enzymes and bioactive metabolites for food supplementation, and provides valuable insights into the metabolomics of human gut microbes.

[0005] Some scholars have conducted a comprehensive assessment of the biosafety of B. elongatus as a probiotic. In addition, studies have reported that oral administration of B. elongatus to mice can significantly improve the obesity symptoms of mice, because B. elongatus produces 12-methylmyristic acid (12-MMA) in mice, which is identified as an important active metabolite of white fat products, has an effective lipid-lowering effect in the body, and activates G protein-coupled receptor 120 (GPR120), thereby stimulating the browning of white adipose tissue.

[0006] However, there is no report on the culture medium and high-density fermentation process of B. elongatus applied to the food industry and the pharmaceutical industry. Although the traditional culture medium can be used to screen and isolate the strain, it is costly, complex in composition, slow in growth rate, and low in viable count, which is not conducive to industrial production. In addition, the traditional culture medium is rich in animal-derived ingredients such as peptone and tryptone, and the products derived from animals may contain prions or contaminants from bacteria, or contain other undesirable ingredients such as allergens, so that it is not suitable for culturing B. elongatus used in human food or medicine.

[0007] Therefore, there is a need in the art for a non-animal-derived, low-cost, high-density culture medium and related fermentation methods for the extended growth of Blautia. Summary of the invention

[0008] Based on the above problems, the purpose of the present invention is to provide a fermentation medium and related fermentation method that are simple in composition, low in cost, and can significantly increase the bacterial mass of Blautia. The inventors of the present invention optimize the fermentation medium and fermentation process for extending Blautia, and combine the fermentation medium with simplified composition and non-animal origin with the control of the pH of the fermentation liquid during the fermentation process and the fed-batch fermentation process, which can significantly increase the bacterial mass of Blautia. Thus, the present invention is completed.

[0009] Therefore, in a first aspect of the present invention, a culture medium for culturing Blautia sp. is provided, which comprises a carbon source, a non-animal nitrogen source, a growth factor, and a prebiotic.

[0010] In a second aspect, a fermentation method of Blautia is provided, which comprises a high-density fermentation step: inoculating a seed liquid of Blautia into the fermentation medium described in the first aspect, and culturing under anaerobic environmental conditions.

[0011] In summary, the present invention provides a high-density culture medium with simple and low-cost ingredients, free of animal-derived ingredients, and capable of efficiently fermenting Blautia, and thereby provides an economical and effective method for culturing Blautia. Specifically, the high-density fermentation culture medium and fermentation method of the present invention are used to culture Blautia, especially extended Blautia, and the bacteria have good growth state, rapid proliferation, short culture time, and good passage stability. In addition, the non-animal-derived culture medium avoids the presence of pollutants or allergens, has higher safety, and can meet the production requirements of the food industry and the pharmaceutical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art will be briefly introduced below.

[0013] Figure 1 The growth curves of Blautia elongata of the present invention under different pH conditions (pH=6.0, 7.0, 8.0, and no pH control) are shown.

[0014] Figure 2 The effect of different inoculum sizes (2%, 6% and 10%) on the growth of Blautia elongata of the present invention in high density fermentation is shown.

[0015] Figure 3 The effect of buffered and unbuffered salts on the growth of Blautia elongata of the present invention is shown.

[0016] Figure 4 The effects of different feeding regimes on the growth of Blautia elongata of the present invention in high-density fermentation are shown.

[0017] Figure 5 The growth curves of Blautia elongata of the present invention at different passage numbers in high-density fermentation are shown.

[0018] Figure 6 The Gram-stained strain morphology of Blautia elongata at different passage numbers is shown.

[0019] Figure 7 The Gram-stained strain morphology of Blautia elongata of the present invention at the first passage is shown.

[0020] Figure 8 A matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) spectrum of Blautia prolonga is shown. DETAILED DESCRIPTION

[0021] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the following description is only used to illustrate the present invention by way of example, and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims. In addition, those skilled in the art will understand that the technical solution of the present invention may be modified without departing from the spirit and purpose of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0023] Where a numerical range is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value to one-tenth of the unit of the lower limit between the upper and lower limits of the range and any other stated or intervening values ​​in the stated range are included in the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and such embodiments are also included in the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or two limits, ranges excluding either or both of those included limits are also included in the subject matter.

[0024] In the context of the present invention, many embodiments use the expressions "comprising", "including" or "consisting essentially / mainly of..." The expressions "comprising", "including" or "consisting essentially / mainly of..." can usually be understood as open-ended expressions, indicating that in addition to the elements, components, assemblies, method steps, etc. specifically listed after the expression, other elements, components, assemblies, method steps, etc. are also included. In addition, in this article, the expressions "comprising", "including" or "consisting essentially / mainly of..." can also be understood as closed expressions in some cases, indicating that only the elements, components, assemblies, method steps specifically listed after the expression are included, and no other elements, components, assemblies, method steps are included. At this time, the expression is equivalent to the expression "consisting of..."

[0025] As commonly used in the art, the term "high-density fermentation" is also known as high-density culture, which refers to a culture technique in which microorganisms grow in a liquid culture in which the cell population density exceeds 10 times that of conventional culture.

[0026] As described above, the present inventors aim to provide a high-density fermentation medium having simple and low-cost ingredients and capable of efficiently fermenting Blautia and a related fermentation method.

[0027] In a first aspect of the present invention, a culture medium for culturing Blautia sp. is provided, which comprises a carbon source, a non-animal nitrogen source, a growth factor, and a prebiotic.

[0028] In a specific embodiment, the Blautia may be Blautia producta.

[0029] In another specific embodiment, the carbon source can be selected from: inorganic carbon sources such as CO2 / H2, CO, CO3 2- 、HCO3 - , organic carbon sources such as glucose, sucrose, maltose, lactose, fructose, trehalose, and any combination thereof.

[0030] In a preferred embodiment, the carbon source is sucrose.

[0031] In a more preferred embodiment, the carbon source is a combination of sucrose and an inorganic carbon source, such as sucrose and HCO3 - combination.

[0032] In yet another specific embodiment, the concentration range of the carbon source may be 2-200 g / L. In a preferred embodiment, the concentration range of the carbon source may be 2-20 g / L.

[0033] In yet another specific embodiment, the non-animal derived nitrogen source can be selected from yeast extract powder, yeast peptone, soy peptone, wheat peptone, and any combination thereof.

[0034] In a preferred embodiment, the non-animal-derived nitrogen source is yeast extract powder or yeast peptone.

[0035] In yet another specific embodiment, the concentration of the non-animal derived nitrogen source may range from 5 to 200 g / L. In a preferred specific embodiment, the concentration of the non-animal derived nitrogen source may range from 5 to 50 g / L.

[0036] In yet another specific embodiment, the growth factor can be selected from cysteine, cysteine ​​hydrochloride and hydrates thereof, hemin, biotin, folic acid, cobalamin, p-aminobenzoic acid, and any combination thereof.

[0037] In a preferred embodiment, the growth factor is cysteine ​​hydrochloride.

[0038] In yet another specific embodiment, the concentration of the growth factor may range from 0.5 to 5 g / L. In a preferred embodiment, the concentration of the growth factor may range from 0.5 to 2 g / L.

[0039] In yet another specific embodiment, the prebiotics may be selected from fructooligosaccharides, xylooligosaccharides, soybean oligosaccharides, inulin, isomaltooligosaccharides, mannooligosaccharides, lactitol, isomaltulose, and any combination thereof.

[0040] In a preferred embodiment, the prebiotic is soybean oligosaccharide.

[0041] In yet another specific embodiment, the concentration of the prebiotics may range from 1 to 50 g / L. In a preferred embodiment, the concentration of the prebiotics may range from 1 to 5 g / L.

[0042] In another specific embodiment, the fermentation medium may further include a buffer salt. In a preferred embodiment, the buffer salt is selected from dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium bicarbonate or any combination thereof, such as a combination of dipotassium hydrogen phosphate, potassium dihydrogen phosphate and sodium bicarbonate, but is not limited thereto. Without wishing to be bound by theory, the inventors have found that buffer salts, especially carbonate-free buffer salts, have little effect on the high-density fermentation of Blautia.

[0043] In yet another specific embodiment, the concentration range of potassium dihydrogen phosphate and dipotassium hydrogen phosphate is 0-4.5 g / L, respectively. In a preferred embodiment, the concentration range of potassium dihydrogen phosphate and dipotassium hydrogen phosphate is 0-1 g / L, respectively.

[0044] In yet another specific embodiment, the concentration range of sodium bicarbonate is 1-10 g / L. In a preferred embodiment, the concentration range of sodium bicarbonate is 1-4 g / L.

[0045] The inventors have found that sodium bicarbonate can be used not only as a buffer salt component but also as an inorganic carbon source in the fermentation medium of the present invention. It is understood that when there is no organic carbon source such as sucrose in the fermentation medium, sodium bicarbonate plays the role of an inorganic carbon source, so that the high-density fermentation of Blautia can still be carried out. In other words, without wishing to be bound by theory, the fermentation medium of the present invention may not include an organic carbon source. However, it is also understood that in the fermentation culture of the present invention, the utilization efficiency of the organic carbon source by the microorganism is higher than that of the inorganic carbon source, and therefore, the organic carbon source is preferred.

[0046] In another specific embodiment, the fermentation medium may include 11.42 g / L sucrose, 21.12 g / L yeast extract, 1.76 g / L soybean oligosaccharides, 1.18 g / L cysteine ​​hydrochloride, and optionally 0.45 g / L dipotassium hydrogen phosphate, 0.45 g / L potassium dihydrogen phosphate and 4 g / L sodium bicarbonate, with the balance being water; or, the fermentation medium includes 21.12 g / L yeast extract, 1.76 g / L soybean oligosaccharides, 1.18 g / L cysteine ​​hydrochloride, 4 g / L sodium bicarbonate, and optionally 0.45 g / L dipotassium hydrogen phosphate and 0.45 g / L potassium dihydrogen phosphate, with the balance being water.

[0047] In a second aspect, a fermentation method of Blautia is provided, which comprises a high-density fermentation step: inoculating a seed liquid of Blautia into the fermentation medium described in the first aspect, and culturing under anaerobic environmental conditions.

[0048] In a specific embodiment, the Blautia is Blautia elongata.

[0049] In yet another specific embodiment, the seed solution is inoculated at an inoculum amount of 2%-10%. In a preferred embodiment, the seed solution is inoculated at an inoculum amount of 2%-4%.

[0050] In yet another specific embodiment, the culturing is carried out at a temperature of 20° C.-40° C. In a preferred embodiment, the culturing is carried out at a temperature of 36° C.-38° C. In a further preferred embodiment, the culturing is carried out at a temperature of 37° C.

[0051] In yet another specific embodiment, the culturing time is 6h-18h.

[0052] In another specific embodiment, the high-density fermentation step further comprises controlling the pH of the fermentation medium during the fermentation process to be between 6 and 8. In a preferred embodiment, the pH of the fermentation medium is controlled to be between 6.0 and 7.5.

[0053] In another specific embodiment, the high-density fermentation step is carried out at a rotation speed of 50-300 rpm. In a preferred embodiment, the high-density fermentation step is carried out at a rotation speed of 50-150 rpm.

[0054] In yet another specific embodiment, the fermentation method further comprises statically culturing the strain to obtain a seed solution of Blautia before the high-density fermentation.

[0055] In a preferred embodiment, the static culture conditions include anaerobic conditions, 36-38° C., 2-3 passages, and a culture time of 12-36 hours for each passage.

[0056] In another specific embodiment, the seed culture medium for static culture can be the same as or different from the high-density fermentation medium, for example, including carbon sources such as glucose, sucrose, maltose, lactose, fructose, trehalose, etc., nitrogen sources such as peptone, tryptone, fish meal, yeast extract powder, yeast peptone, soy peptone, wheat peptone, etc., growth factors such as cysteine, cysteine ​​hydrochloride and its hydrate, hemin chloride, biotin, folic acid, cobalamin, p-aminobenzoic acid, etc., prebiotics such as fructooligosaccharides, xylooligosaccharides, soybean oligosaccharides, inulin, isomaltooligosaccharides, mannooligosaccharides, lactitol, isomaltulose, etc., and buffer salts such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and sodium bicarbonate.

[0057] In an exemplary embodiment, the seed culture medium for static culture includes: 5 g / L glucose, 10 g / L trypticase, 2.5 g / L yeast extract, 0.45 g / L dipotassium hydrogen phosphate, 0.45 g / L potassium dihydrogen phosphate, 0.9 g / L NaCl, 0.44 g / L MgSO4·7H2O, 0.99 g / L CaCl2, 4 g / L sodium bicarbonate, and the balance is water.

[0058] In another exemplary embodiment, the static culture seed culture medium may include: 11.42 g / L sucrose, 21.12 g / L yeast extract, 1.76 g / L soybean oligosaccharides, 1.18 g / L cysteine ​​hydrochloride, 0.45 g / L dipotassium hydrogen phosphate, 0.45 g / L potassium dihydrogen phosphate, 4 g / L sodium bicarbonate, and the balance is water.

[0059] In another specific embodiment, the high-density fermentation is carried out in a fed-batch fermentation mode. For example, a slow continuous feeding mode can be used for feeding during the high-density fermentation process. As commonly used in the art, the term "fed-batch fermentation" refers to a fermentation technique in which a certain material is added to the fermentation system in a certain manner during the batch fermentation of microorganisms, but the fermentation liquid is not continuously discharged to the outside. This fermentation technique is a fermentation technique between batch fermentation and continuous fermentation. According to whether the feeding time is continuous, it can be divided into intermittent feeding and continuous feeding. Those skilled in the art will know that a high concentration of substrate in the culture medium will inhibit the growth of microorganisms, while fed-batch fermentation is first fermented under a relatively low nutrient concentration, and then fresh nutrients are continuously added to allow the bacteria to grow normally and continuously accumulate metabolites, thereby avoiding substrate inhibition caused by excessive one-time feeding in batch fermentation.

[0060] In another specific embodiment, the feed can be a complete feed medium or a semi-batch feed medium. The so-called "complete feed medium" refers to a medium supplemented with a complete medium, while the so-called "semi-batch feed medium" refers to a medium supplemented with only one or several nutrients, for example, only an incomplete medium including a carbon source such as sucrose, or an incomplete medium including a carbon source such as sucrose and a prebiotic such as soybean oligosaccharide.

[0061] In another preferred embodiment, during high-density fermentation, a feed flow rate of 2 g·L -1 ·h -1 To 6g·L -1 ·h -1 The total sugar concentration of the feed medium can be 100-300 g / L.

[0062] In the context of the present invention, the term "total sugar" refers to the sum of all sugar substances contained in the culture medium. Thus, the term "total sugar rate" refers to the sum of the flow rates of all sugar substances added during the feed process; the term "total sugar concentration" refers to the concentration of all sugar substances contained in the culture medium.

[0063] Those skilled in the art will know that, for the present invention, based on the use of the fermentation medium optimized by the inventors, one or more nutrients such as carbon sources required for the fermentation of Blautia are intermittently added, and the bacteria can grow and reproduce in large quantities without interruption, thereby achieving high-density culture of Blautia, and avoiding the problem of decreased bacterial volume due to the extension of the centrifugation stage after the fermentation. In addition, the use of the fed-batch method can further save fermentation time, shorten the fermentation cycle, improve fermentation efficiency, simplify the fermentation steps, reduce equipment investment and reduce production costs, which is very beneficial for the large-scale production of Blautia.

[0064] Biological sample preservation information: Blautia producta strain BWT1 was deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Administration on June 24, 2019, with the deposit number CGMCCNO.18016; the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It was detected as a viable strain by the collection center on June 24, 2019.

[0065] The beneficial effects of the present invention are as follows: the raw materials used in the present invention can all be of non-animal origin, thus avoiding the presence of ingredients in animal-derived products that do not comply with food and drug regulations. The culture medium of the present invention has simple ingredients, with only 7 ingredients in the seed culture medium used for static culture, and only 4 ingredients in the fermentation culture medium used for high-density fermentation to achieve a very high number of viable bacteria, while the traditional culture medium has more than 20 ingredients. Compared with the traditional culture medium, the number of viable fermentation bacteria obtained by the method of the present invention is increased by one order of magnitude. The non-animal-derived culture medium of the present invention has low cost, simple industrial production operation, high number of viable fermentation bacteria, high production efficiency, good economic benefits, and is easy to realize the industrial production of extended Blautia in the food industry and the pharmaceutical industry.

[0066] Hereinafter, the present invention will be described in more detail in conjunction with exemplary embodiments. However, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be considered as intended to interpret the scope of the present invention.

[0067] Example

[0068] The high-density culture method and related characterization of the extended Blautia of the present invention are given in the following examples. Unless otherwise specified, the test methods used therein are all conventional methods, and unless otherwise specified, the test materials used in the following examples are all purchased from conventional reagent stores. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention.

[0069] It should be noted that the terms used in the specification of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The above summary of the invention and the detailed description below are only for the purpose of specifically explaining the present invention, and are not intended to limit the present invention in any way. Without departing from the spirit and purpose of the present invention, the scope of the present invention is determined by the attached claims.

[0070] Example 1: Effects of different carbon sources on the growth of Blautia prolongata

[0071] Different carbon source culture media were prepared as secondary culture media according to the ingredients shown in formulas 1-5 given in Table 1 below, among which formula 1 containing glucose was also used as primary culture media. Take out the cryotubes from the -80°C refrigerator and thaw at room temperature, inoculate them into the primary culture media at a 2% inoculation rate in an anaerobic chamber, and obtain the primary seed solution after culturing for 24 hours. Inoculate it into the secondary culture media containing different carbon sources shown in formulas 1-5 at a 2% inoculation rate to start culturing, and obtain the secondary seed solution. Anaerobic chamber (Whitley 35, UK) culture conditions: anaerobic (N2:H2=90:10), 37°C. After culturing for 24 hours, OD600 was detected with an enzyme marker (Tecan, Switzerland), and the results are also shown in Table 1 below.

[0072] From the OD600 results in Table 1, it can be seen that the secondary culture medium containing sucrose (Formulation 4) obtained the highest OD600. Therefore, sucrose is a preferred carbon source for prolonging the growth of Blautia.

[0073] Table 1: Effects of different culture media on the growth of Blautia

[0074]

[0075] Example 2: Effects of different prebiotics on the growth of Blautia

[0076] According to the ingredients shown in formula 1-3 given in Table 2 below, a secondary culture medium containing different prebiotic ingredients was prepared, wherein the primary culture medium was the same as the primary culture medium in Example 1. A cryovial was taken out of a -80°C refrigerator and thawed at room temperature, and inoculated into the primary culture medium at a 2% inoculum in an anaerobic chamber. After culturing for 24 hours, a primary seed solution was obtained, which was inoculated into the secondary culture medium shown in formula 1-3 at a 2% inoculum to start culturing, and a secondary seed solution was obtained. Anaerobic chamber (Whitley 35, UK) culture conditions: anaerobic (N2:H2=90:10), 37°C. After culturing for 24 hours, OD600 was detected using a microplate reader (Tecan, Switzerland), and the results are also shown in Table 2 below.

[0077] From the OD600 results in Table 2, it can be seen that the secondary culture medium containing soybean oligosaccharides as prebiotics (Formula 2) obtained the highest OD600. Therefore, soybean oligosaccharides are more suitable for prolonging the growth of Blautia.

[0078] Table 2: Effects of different prebiotics on the growth of Blautia

[0079]

[0080]

[0081] Example 3: Comparison of non-animal-derived culture medium and animal-derived culture medium

[0082] According to the ingredients shown in formulas 1-5 given in Table 3 below, secondary culture media with non-animal and animal sources were prepared, respectively, wherein the primary culture medium was the same as the primary culture medium in Example 1. Take out the cryotube from the -80°C refrigerator and thaw it at room temperature, inoculate it into the primary culture medium at a 2% inoculation rate in an anaerobic box, and obtain a primary seed solution after culturing for 24 hours. Inoculate it into the secondary culture medium in Table 3 at a 2% inoculation rate to obtain a secondary seed solution. Anaerobic box (Whitley 35, UK) culture conditions: anaerobic (N2:H2=90:10), 37°C. After culturing for 24 hours, use an ELISA reader (Tecan, Switzerland) to detect OD600 and perform viable counts. The results are also shown in Table 3 below.

[0083] Table 3: Comparison of non-animal-derived culture media and animal-derived culture media

[0084]

[0085] As shown in Table 3, in the prior art, a combination of yeast extract powder and tryptone is often used as a nitrogen source (Formula 1). However, after separating them, the OD600 of yeast extract powder alone as a nitrogen source (Formula 2) is not significantly different from that of Formula 1 (p>0.05). However, the OD600 of tryptone alone as a nitrogen source (Formula 4) is much lower than that of Formula 1. In addition, the OD600 of non-animal nitrogen source yeast peptone (Formula 3) is not significantly different from that of Formula 1. In addition, in Formula 5, peptone is used as a nitrogen source, and the OD600 is the lowest. The prices of yeast extract powder and yeast peptone are both lower than those of tryptone.

[0086] Example 4: Comparison of the culture medium of the present invention with the original culture medium

[0087] In this embodiment, a secondary culture medium was prepared according to Table 4 below, wherein Formula 1 is the original culture medium formula given in the literature (Duncan SH, Hold GL, Harmsen HJM, et al. Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacteriumprausnitzii gen.nov., comb.nov[J]. International journal of systematic and evolutionary microbiology, 2002, 52(6): 2141-2146), and Formula 2 is the formula of the culture medium of the present invention.

[0088] Take the cryopreserved tubes from the -80℃ refrigerator and thaw at room temperature. Inoculate the primary culture medium at 2% in an anaerobic box. After culturing for 24 hours, obtain the primary seed solution, which is inoculated into the secondary culture medium at 2% inoculation to obtain the secondary seed solution. The 8 vitamin components and their addition amounts in the original culture medium are shown in the literature of Duncan SH et al. Anaerobic box (Whitley 35, UK) culture conditions: anaerobic (N2:H2=90:10), 37℃. After culturing for 24 hours, use a microplate reader (Tecan, Switzerland) to detect OD600 and count the viable bacteria. The results are also shown in Table 4.

[0089] Table 4: Comparison of fermentation effects between the culture medium of the present invention and the original culture medium

[0090]

[0091]

[0092] From the results in Table 4, it can be seen that compared with Formula 1, Formula 2 of the present invention has an OD600 increase of more than 190% and a viable bacterial count increase of about 24 times.

[0093] Example 5: Effect of pH on the extension of high-density fermentation culture of Blautia

[0094] In this example, the preparation of the primary culture medium and the primary seed solution is the same as in Example 1. The secondary culture medium is the same as the culture medium of Formula 2 in Example 4. After the primary seed solution of Blautia prolonga is cultured in the secondary culture medium for 24 hours, it is inoculated in a 5L fermenter (containing 3L fermentation medium) for high-density fermentation, with an inoculation amount of 2%. The high-density fermentation medium is the same as the secondary culture medium.

[0095] High-density fermentation conditions: 37°C, anaerobic (N2:H2:CO2=85:10:5), 150rpm. The pH was adjusted to 8.0, 7.0, 6.0 and pH uncontrolled by alkali solution to explore the effects of different pH and uncontrolled pH on the extension of Blautia high-density fermentation.

[0096] Depend on Figure 1 It can be seen that under uncontrolled pH conditions, the logarithmic growth phase started 8 hours after inoculation, and continued for 6 hours until the stable phase was reached at 14 hours. Under pH 8 conditions, the logarithmic growth phase started 10 hours after inoculation, and continued for 6 hours to 16 hours before reaching the stable phase. Under pH 6 and pH 7 conditions, logarithmic growth started 6 hours after inoculation, and fermentation reached the stable phase at 12 hours, with OD600 reaching the highest under pH 6.0 conditions.

[0097] Example 6: Effect of different inoculum sizes on the growth of Blautia

[0098] Prepare the primary and secondary seed solutions and high-density fermentation medium according to Example 5.

[0099] High-density fermentation conditions: fermentation pH 6.5, 37°C, anaerobic (N2:H2:CO2=85:10:5), 150rpm, inoculation amounts of 2%, 6%, and 10%, respectively, to explore the effects of different inoculation conditions on the extension of high-density fermentation of Blautia.

[0100] The changes of OD600 over time during high-density culture are shown in Figure 2. Figure 2 As shown. Figure 2 It can be seen that the increase in inoculation amount has no significant effect on the start of the logarithmic growth phase, and has limited effect on the OD600 at the end of fermentation. The OD600 curves under various inoculation amount conditions have a high degree of overlap. In addition, when the inoculation amount is 2%, the OD600 is the highest when the fermentation is 12h. Increasing the inoculation amount to 6% and 10% will reduce the OD600 at the end of fermentation to a certain extent.

[0101] Example 7: Effect of buffer salt on the extension of high-density fermentation of Blautia

[0102] Prepare the primary and secondary seed solutions according to Example 5.

[0103] Two groups of high-density fermentation medium were prepared:

[0104] 1) Buffer salt group: sucrose 11.42 g / L, yeast extract powder 21.12 g / L, soybean oligosaccharide 1.76 g / L, cysteine ​​hydrochloride 1.18 g / L, dipotassium hydrogen phosphate 0.45 g / L, potassium dihydrogen phosphate 0.45 g / L, sodium bicarbonate 4 g / L;

[0105] 2) Non-buffered salt group: sucrose 11.42 g / L, yeast extract 21.12 g / L, soybean oligosaccharide 1.76 g / L, cysteine ​​hydrochloride 1.18 g / L.

[0106] The changes of OD600 over time during high-density fermentation culture are shown in Figure 2. Figure 3 As shown. Figure 3 It can be seen that after removing the buffer salt from the culture medium, the growth curve of the non-buffer salt group and the growth curve of the buffer salt group had a high degree of overlap. When fermented to the highest OD600 point, although the OD600 value of the non-buffer salt group was lower than that of the buffer salt group, the difference was not significant (p>0.05).

[0107] Example 8: Effect of fed-batch fermentation on the extension of high-density fermentation of Blautia

[0108] The primary and secondary seed solutions were prepared according to Example 5, and the high-density fermentation medium was prepared according to the buffer-free salt group of Example 7.

[0109] High-density fermentation conditions: pH 6.5, 37°C, anaerobic (N2:H2:CO2=85:10:5), 150rpm, inoculation amount 4%. Feeding began after growth to the stable phase.

[0110] Semi-batch fed medium: a mixed solution of sucrose and soybean oligosaccharides (the concentrations of sucrose and soybean oligosaccharides are 10 times the corresponding concentrations in formula 2 of Example 4). There are two feeding methods:

[0111] Group A: 5.42 g·L -1 ·h -1 The total sugar rate was added into the semi-fed-batch medium;

[0112] Group B: 2.71 g·L -1 ·h -1 The total sugar rate was added into the semi-batch fed medium.

[0113] The batch feeding was continued until the stable phase was reached again. The growth of Blautiae was compared under the two feeding methods. The results are shown in Figure 4 .Depend on Figure 4 It can be seen that in the non-fed group, OD600 reached the highest point and then rapidly decreased with the extension of culture time. At the two feeding rates, the growth of Blautiae in the fed group was similar, and neither of them could obtain a higher OD600, but it could be kept constant. The addition of semi-batch fed medium can solve the problem of decreased bacterial mass caused by the extension of the centrifugation stage after the end of fermentation.

[0114] Example 9: Study on the stability of extended passage of Blautia

[0115] The first generation of Blautia extensa was inoculated in the primary culture medium prepared according to Example 5. In this experiment, the subsequent subculture culture medium was divided into two groups:

[0116] Group A: The culture medium for passages 2-30 was the same as the primary culture medium prepared according to Example 5;

[0117] Group B passage: 2-30 passages The culture medium was the same as the secondary culture medium prepared according to Example 5.

[0118] The inoculum volume was 1% (v / v), and the cells were subcultured every 24 hours for 30 generations. OD600 was tested at each generation (the results were as follows: Figure 5 The results are shown in Table 1), and the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) identification spectrum was obtained by Autof ms1000 (Antu Biotechnology, China) (the results are shown in Table 1). Figure 8 The strain morphology was examined by microscopic examination at the 1st, 10th, 20th and 30th generations (the results are shown in Figure 6 , Figure 7 shown).

[0119] Depend on Figure 5 It can be seen that the OD600 of Blautia in the culture medium of group B was more stable and the growth was more stable than that in the culture medium of group A.

[0120] Figure 6 and Figure 7 The Gram-stained strain morphology of Blautia elongata at the 10th, 20th and 30th passages and the first passage is shown respectively. Blautia elongata is spherical or oval in shape and appears in pairs or chains. Figure 6 It can be seen that when the extended Blautia was subcultured in the two culture media, the strain morphology was consistent with the above description and the state was stable without abnormal changes. In group A, the cells were more dispersed and mostly appeared in pairs. In group B, the cells appeared in chains, which was similar to Figure 7 The status is closer.

[0121] Figure 8 The matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) spectrum of Blautia prolonga is shown. Figure 8 It can be seen that the highest score of this map is 9.646, with species level confidence, and it is identified as Blautia producta. (Score explanation: [9.5, 10] species level confidence, possibly a subspecies; [9.0, 9.5) species level confidence; [6.0, 9.0) genus level confidence; [0.0, 6.0) unconfident).

Claims

1. A method for culturing Blautia extensa ( Blautia producta ) of the fermentation medium, which comprises: A carbon source with a concentration range of 2-20 g / L, wherein the carbon source is sucrose, or sucrose and HCO3 - A combination of Yeast extract powder with a concentration range of 5-50 g / L, A growth factor in a concentration range of 0.5-2 g / L selected from cysteine ​​or cysteine ​​hydrochloride and hydrates thereof, Soybean oligosaccharides in a concentration range of 1-5 g / L.

2. The fermentation medium according to claim 1, wherein The growth factor is cysteine ​​hydrochloride.

3. The fermentation medium according to claim 1, wherein The fermentation medium further comprises a buffer salt selected from dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium bicarbonate or any combination thereof.

4. The fermentation medium according to claim 3, wherein The concentration ranges of the potassium dihydrogen phosphate and dipotassium hydrogen phosphate are respectively 0-4.5 g / L; the concentration range of the sodium bicarbonate is 1-10 g / L.

5. The fermentation medium according to claim 4, wherein The concentration ranges of the potassium dihydrogen phosphate and dipotassium hydrogen phosphate are respectively 0-1 g / L; the concentration range of the sodium bicarbonate is 1-4 g / L.

6. The fermentation medium according to any one of claims 1 to 5, wherein The fermentation medium comprises 11.42 g / L sucrose, 21.12 g / L yeast extract, 1.76 g / L soybean oligosaccharides, 1.18 g / L cysteine ​​hydrochloride, and optionally 0.45 g / L dipotassium hydrogen phosphate, 0.45 g / L potassium dihydrogen phosphate and 4 g / L sodium bicarbonate, with the balance being water; or, the fermentation medium comprises 11.42 g / L sucrose, 21.12 g / L yeast extract, 1.76 g / L soybean oligosaccharides, 1.18 g / L cysteine ​​hydrochloride, 4 g / L sodium bicarbonate, and optionally 0.45 g / L dipotassium hydrogen phosphate and 0.45 g / L potassium dihydrogen phosphate, with the balance being water.

7. A fermentation method of Blautia elongata, comprising a high-density fermentation step: inoculating a seed solution of Blautia elongata into a fermentation medium according to any one of claims 1 to 6 at an inoculation rate of 2% to 10%, and culturing under anaerobic conditions at a temperature of 20° C. to 40° C. for 6 h to 18 h; wherein, The pH of the fermentation medium during the fermentation process was controlled at 6-8.

8. The method according to claim 7, wherein: The temperature is 36°C-38°C.

9. The method according to claim 7 or 8, wherein: The seed liquid is inoculated at an inoculation rate of 2%-4%.

10. The method according to claim 7 or 8, wherein: The pH of the fermentation medium is controlled to be 6.0-7.

5.

11. The method according to claim 7 or 8, wherein: The high-density fermentation step is carried out at a rotation speed of 50-300 rpm.

12. The method according to claim 7 or 8, wherein: The high-density fermentation step is carried out at a rotation speed of 50-150 rpm.

13. The method according to claim 7 or 8, further comprising statically culturing the strain to obtain a seed solution of Blautia elongata before the high-density fermentation.

14. The method according to claim 13, wherein: The static culture conditions include anaerobic conditions, 36-38° C., 2-3 passages, and a culture time of 12-36 hours for each passage.

15. The method according to claim 7 or 8, wherein: The high-density fermentation adopts a fed-batch fermentation method.

16. The method according to claim 15, wherein: The feed is a complete fed medium or a semi-fed batch medium.

17. The method according to claim 15, wherein: The feed uses an incomplete culture medium comprising a carbon source and soybean oligosaccharides.

18. The method according to claim 15, wherein: The feed flow rate is 2-6 g·L -1 •h -1 Slow continuous feeding at a total sugar rate of 100%.

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