Method for improving fermentation intensity and spore transformation of clostridium butyricum

Abstract

The invention relates to the field of microbial fermentation, and discloses a method for improving the fermentation intensity and spore transformation of clostridium butyricum, which comprises the following steps: inoculating clostridium butyricum into a fermentation culture medium for fermentation culture; supplementing an inorganic ion source into the fermentation liquid at the end of logarithmic growth of the fermentation culture, and continuing the fermentation culture until the spore rate is not lower than 90%; then, optionally, a protective agent is supplemented into the fermentation liquid for induction culture, so that the spore rate is further increased; the initial osmotic pressure of the fermentation culture medium under 0.05 MPa is less than or equal to 460 mOsm / kg; the inorganic ion source is selected from at least one of a potassium ion source, a manganese ion source and a calcium ion source. According to the method, the fermentation intensity in the early stage of clostridium butyricum fermentation and the spore conversion rate in the middle and later stages can be improved, and the fermentation period is shortened; spore transformation can be further improved, completely mature spores are obtained, and the performance of the spores is improved; meanwhile, the content of short-chain fatty acid in the fermentation liquor is greatly increased.

Description

Technical Field

[0001] The invention relates to the field of microbial fermentation, and in particular to a method for improving the fermentation intensity and spore conversion rate of Clostridium butyricum. Background Art

[0002] Clostridium butyricum is a strict anaerobic bacterium, mainly found in cheese, natural yogurt, healthy animal intestines, animal feces and soil. Its metabolites mainly include butyric acid, acetic acid, lactic acid, etc. The cells in PYG broth are straight rods with blunt ends, motile and peritrichous flagella, 0.5-1.7μm×2.4-7.6μm. Single, paired or short chain, occasionally filamentous bacteria, elliptical spores, mesogenic or subterminal, generally do not cause cell expansion. Clostridium butyricum exists in an endogenous spore state, has strong tolerance to low gastric acid, bile resistance, strong resistance to the external environment, and can still maintain a high activity and viable count after high-temperature processing and granulation.

[0003] The low fermentation level and spore conversion rate of existing Clostridium butyricum have become bottleneck problems in the fermentation production of Clostridium butyricum. In order to further improve the fermentation intensity and spore conversion rate of Clostridium butyricum, the existing high-density fermentation technology of Clostridium butyricum often achieves high-density fermentation by optimizing the addition of various carbon and nitrogen sources and inorganic salts in the fermentation medium; or by adding inducers in the late fermentation stage to achieve the purpose of increasing the spore rate.

[0004] CN116590212A discloses a method for improving the spore conversion rate of Clostridium butyricum, which comprises: inoculating Clostridium butyricum in a seed culture medium for culturing, and performing water bath treatment after the culturing is completed to obtain a seed solution; taking the seed solution and inoculating it into a fermentation culture medium for fermentation, adding supplementary materials during the fermentation, and maintaining the pH at 6.5-7.0 with an alkali solution; after the supplementary materials are completed, maintaining the pH at 6.5-7.0 with a CaCO3 turbid solution until the fermentation is completed. The method improves the spore conversion rate of Clostridium butyricum by adding a nitrogen source and a carbon source during the fermentation, adjusting the pH with an alkali solution, and adjusting the pH with CaCO3 in the later stage until the fermentation is completed.

[0005] CN114214263A discloses a method for promoting spore formation of Clostridium butyricum and its microecological preparation and application. The method comprises adding a spore production inducer in the middle stage of fermentation culture, wherein the components of the spore production inducer include inactivated Clostridium butyricum mud, 2,6-pyridinedicarboxylic acid and calcium carbonate. The method effectively increases the amount of live Clostridium butyricum and the spore rate. However, sodium alginate and agar as thickeners and ferrous sulfate as a reducing agent are also added to the fermentation medium to block oxygen, and the composition of the medium is complex. The inactivated Clostridium butyricum mud also needs to be obtained through induction culture, inactivation, and solid-liquid separation, and the preparation method is complicated.

[0006] In summary, although the above method improves the bacterial concentration and spore rate, the operation is relatively cumbersome, the production cost is high and the efficiency is low; secondly, the fermentation intensity of Clostridium butyricum is not high in the early stage, and the fermentation cycle is long; the spore conversion in the middle and late stages is limited, and the spore conversion rate at the fermentation end point cannot be further improved. The heat resistance and gastric acid and bile resistance of the spores are limited, and the content of short-chain fatty acids (especially acetic acid and butyric acid) in the fermentation broth is low. Summary of the invention

[0007] The purpose of the present invention is to overcome the problems of long fermentation period of Clostridium butyricum, low fermentation intensity and spore conversion, and poor spore performance in the prior art, and to provide a preparation method for improving the fermentation intensity and spore conversion rate of Clostridium butyricum. The method can improve the fermentation intensity of Clostridium butyricum in the early stage of fermentation and the conversion rate of spores in the middle and late stages, shorten the fermentation period; improve the heat resistance and gastric acid and bile salt resistance of the spores; and also can greatly increase the content of short-chain fatty acids (especially acetic acid and butyric acid) in the fermentation broth.

[0008] The inventors of the present invention have found that optimizing the initial osmotic pressure of the fermentation medium and supplementing an inorganic ion source at the end of the logarithmic growth period can effectively promote the proliferation and spore production of Clostridium butyricum, and achieve high-density fermentation and high spore conversion rate; adding a protective agent after the fermentation tank is stopped can promote the spore rate to further increase to 100%, obtain fully mature spores, and improve the heat resistance and gastric acid and bile resistance of the spores; in addition, the content of short-chain fatty acids (especially acetic acid and butyric acid) in the fermentation broth is also significantly improved.

[0009] In order to achieve the above object, the present invention provides a preparation method for improving the fermentation intensity and spore conversion of Clostridium butyricum, the method comprising: inoculating Clostridium butyricum into a fermentation medium for fermentation culture; adding an inorganic ion source to the fermentation broth at the end of the logarithmic growth period of the fermentation culture to continue the fermentation culture until the spore rate is not less than 90%; then, optionally, adding a protective agent to the fermentation broth for induction culture to further improve the spore rate;

[0010] Wherein, the initial osmotic pressure of the fermentation medium at 0.05 MPa is ≤460 mOsm / kg;

[0011] Wherein, the inorganic ion source is selected from at least one of a potassium ion source, a manganese ion source and a calcium ion source.

[0012] Through the above technical solution, the present invention can at least achieve the following beneficial technical effects:

[0013] (1) The method of the present invention can effectively improve the fermentation intensity of Clostridium butyricum (i.e., the number of Clostridium butyricum increased per unit time, 10 8 CFU / (mL . h));

[0014] (2) The method of the present invention has a high spore conversion rate for Clostridium butyricum, and the spore conversion rate can be further improved after the fermentation is completed. In a preferred embodiment, the spore conversion rate of Clostridium butyricum in the middle and late stages of fermentation is more than 97%, and the spore conversion rate is further increased to 100% after the fermentation is completed;

[0015] (3) The method of the present invention can shorten the fermentation cycle and save production costs;

[0016] (4) The method of the present invention increases the content of short-chain fatty acids (especially acetic acid and butyric acid) in the fermentation broth;

[0017] (5) The method of the present invention can obtain fully mature spores, which have better heat resistance and gastric acid and bile salt resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a curve diagram showing the change of the live bacterial count of Clostridium butyricum in the fermentation broth of the embodiment and the comparative example with the fermentation time.

[0019] Figure 2 It is a curve diagram showing the change of spore rate of Clostridium butyricum in the fermentation broth of the embodiment and the comparative example with the fermentation time. DETAILED DESCRIPTION

[0020] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0021] The present invention provides a preparation method for improving the fermentation intensity and spore conversion of Clostridium butyricum, which comprises: inoculating Clostridium butyricum into a fermentation medium for fermentation culture; adding an inorganic ion source to the fermentation liquid at the end of the logarithmic growth period of the fermentation culture to continue the fermentation culture until the spore rate is not less than 90% (such as 92%-95%); then, optionally, adding a protective agent to the fermentation liquid for induction culture to further improve the spore rate;

[0022] Wherein, the initial osmotic pressure of the fermentation medium at 0.05 MPa is ≤460 mOsm / kg;

[0023] Wherein, the inorganic ion source is selected from at least one of a potassium ion source, a manganese ion source and a calcium ion source.

[0024] According to some embodiments of the present invention, the addition amount of the inorganic ion source is 1.45-4.6 g / L based on the total volume of the fermentation medium.

[0025] In the present invention, the potassium ion source may be a substance commonly used in the art that can be dissolved in water and provide potassium ions. Preferably, the potassium ion source is selected from potassium dihydrogen phosphate and / or dipotassium hydrogen phosphate.

[0026] In the present invention, the manganese ion source may be a substance commonly used in the art that can dissolve in water and provide manganese ions. Preferably, the manganese ion source is selected from manganese sulfate and / or manganese chloride.

[0027] In the present invention, the calcium ion source may be a substance commonly used in the art that can provide calcium ions. Preferably, the calcium ion source is selected from calcium chloride and / or calcium carbonate.

[0028] In the present invention, the inorganic ion source can be added to the fermentation broth in the form of an inorganic ion source solution. Preferably, the addition amount of the inorganic ion source solution is 5v%-10v% of the volume of the fermentation medium.

[0029] Preferably, the inorganic ion source solution is added within 2 h (eg, 0.5 h, 1 h, 1.5 h or 2 h).

[0030] In the present invention, the fermentation culture of Clostridium butyricum occurs continuously, and the fermentation of Clostridium butyricum is always ongoing from the start to the end of the inorganic ion source supplementation, that is, the duration of the inorganic ion source supplementation means the duration of the fermentation culture.

[0031] According to some embodiments of the present invention, preferably, the initial osmotic pressure of the fermentation medium at 0.05 MPa is 400-440 mOsm / kg.

[0032] According to some embodiments of the present invention, the supplementation time of the inorganic ion source (ie, the time when Clostridium butyricum reaches the end of the logarithmic growth phase) is 12-16 hours after inoculation, preferably 12-14 hours.

[0033] According to some embodiments of the present invention, based on the total volume of the fermentation medium, the inoculation amount of Clostridium butyricum is 5×10 8 -1.5×10 9 CFU / L.

[0034] In the present invention, the protective agent may be a substance commonly used in the art that can induce Clostridium butyricum to transform into spores. According to a preferred embodiment of the present invention, the protective agent is selected from at least one of potassium salt, calcium salt and Lactobacillus plantarum.

[0035] Preferably, the potassium salt is selected from potassium dihydrogen phosphate and / or dipotassium hydrogen phosphate.

[0036] Preferably, the strain preservation number of the Lactobacillus plantarum is CGMCC No.15013 (disclosed in CN109423467A).

[0037] In the present invention, the potassium salt and calcium salt in the protective agent can be added in the form of an inorganic salt solution; and the Lactobacillus plantarum is provided in the form of a Lactobacillus plantarum fermentation liquid.

[0038] More preferably, the method for preparing the Lactobacillus plantarum fermentation broth comprises: 9 -9×10 9 The Lactobacillus plantarum was inoculated into the MRS medium at an inoculation amount of CFU / L, and cultured at 37±2°C for 16-20h to obtain the Lactobacillus plantarum fermentation liquid.

[0039] According to some embodiments of the present invention, the protective agent is potassium salt, and the added amount of the protective agent (potassium salt) is 2-25 g / L, preferably 5-12 g / L, based on the total volume of the fermentation medium.

[0040] According to some embodiments of the present invention, the protective agent is a calcium salt, and the added amount of the protective agent (calcium salt) is 2-20 g / L, preferably 8-14 g / L, based on the total volume of the fermentation medium.

[0041] According to some embodiments of the present invention, the protective agent is Lactobacillus plantarum, and the amount of the protective agent (Lactobacillus plantarum) added is 1×10 11 -9.9×10 11 CFU / L, preferably 2×10 11 -7.5×10 11 CFU / L.

[0042] In the present invention, the inoculation amount of the Lactobacillus plantarum fermentation broth is 5v%-15v% of the volume of the fermentation medium.

[0043] In the present invention, the protective agent is added after the fermentation is completed, that is, when the number of live bacteria in the fermentation liquid no longer changes significantly at the end of the logarithmic growth period and the spore rate under microscopic examination reaches more than 94%, the fermentation is terminated by placing the tank, and the protective agent is added to the fermentation liquid for induction culture to promote further transformation of the spores.

[0044] According to some embodiments of the present invention, the fermentation medium comprises a carbon source, a nitrogen source, a magnesium salt, Tween and water.

[0045] Preferably, the fermentation medium comprises corn flour, glucose, soluble starch, yeast extract powder, peptone, magnesium sulfate, Tween 80, amino acids and water.

[0046] More preferably, the amino acids include lysine, glutamic acid and methionine.

[0047] More preferably, the mass ratio of lysine, glutamic acid and methionine in the amino acids is 1:

[0048] (0.5-1.0):(0.5-1.0).

[0049] In the present invention, lysine, glutamic acid and methionine were all purchased from Tmall, and the product models were 98.5% lysine hydrochloride, L-glutamic acid (food grade), and DL-methionine, respectively.

[0050] According to some embodiments of the present invention, based on the total volume of the fermentation medium, the content of the corn flour is 10-30 g / L, the content of the glucose is 20-45 g / L, the content of the soluble starch is 5-15 g / L, the content of the yeast extract is 10-15 g / L, the content of the peptone is 15-20 g / L, the content of the magnesium sulfate is 0.2-1 g / L, the content of the Tween 80 is 0.5-2 g / L, the content of the amino acid is 2-4 g / L, and the balance is water.

[0051] In the present invention, the Clostridium butyricum refers to Clostridium butyricum after activation treatment. It should be understood that the activation method can be a conventional method used in the art, for example, the glycerol bacteria containing Clostridium butyricum stored in an ultra-low temperature refrigerator (such as -80°C) can be naturally thawed and then inoculated into a liquid culture medium for seed culture.

[0052] According to some embodiments of the present invention, the method further comprises: before inoculating into the fermentation medium, inoculating Clostridium butyricum glycerol bacteria into a seed medium for seed culture to obtain a seed solution containing Clostridium butyricum (for inoculation into the fermentation medium).

[0053] According to some embodiments of the present invention, the conditions for seed culture include: temperature of 36-38°C, time of 14-20h, pH of 5.2-7, and anaerobic culture.

[0054] Preferably, the seed culture medium is RCM culture medium.

[0055] Preferably, the inoculation amount of Clostridium butyricum is 1-3v‰ of the volume of the seed culture medium.

[0056] In the present invention, the type of Clostridium butyricum is not particularly limited, and may be Clostridium butyricum conventionally used in the art. For example, the Clostridium butyricum used in the present invention is derived from COFCO Biotech Co., Ltd., and its strain collection number is CGMCC No. 20417 (disclosed in CN112980735A).

[0057] In the present invention, the seed culture can be carried out in an anaerobic bottle, and those skilled in the art can adjust the specifications of the anaerobic bottle according to actual conditions.

[0058] Preferably, the liquid volume of the seed culture medium is 40-70v%.

[0059] Preferably, the inoculation amount of Clostridium butyricum is 1-3v‰ of the volume of the seed culture medium.

[0060] According to some embodiments of the present invention, the conditions of the fermentation culture (culture after inoculation and continued fermentation culture with addition of inorganic ion source) include: temperature of 36-38°C, pH of 6-7, and anaerobic culture.

[0061] In the present invention, the fermentation culture process is divided into the early fermentation culture period (0-12h) and the logarithmic growth period (12-18h).

[0062] According to some embodiments of the present invention, the induction culture conditions include: a temperature of 36-38° C. and a time of 12-24 h.

[0063] In the present invention, the fermentation culture can be carried out in a fermenter, and those skilled in the art can adjust the specifications of the fermenter according to actual conditions, and can also adjust the stirring speed.

[0064] Preferably, the volume of the fermentation medium is 70v%-80v%.

[0065] Preferably, the stirring speed is 100-300 rpm.

[0066] In the present invention, the fermentation culture of Clostridium butyricum is carried out in an anaerobic environment. In order to maintain the anaerobic environment of the fermentation tank, a small amount of sterile nitrogen can be introduced before fermentation to maintain a certain pressure.

[0067] Preferably, the ventilation volume of the nitrogen is 0.1-0.2 vvm; the pressure is 0.01 MPa-0.05 MPa.

[0068] According to a particularly preferred embodiment of the present invention, there is provided a preparation method for improving the fermentation intensity and spore transformation of Clostridium butyricum, the method comprising:

[0069] (1) The seed culture medium is RCM medium;

[0070] (2) the fermentation medium comprises a carbon source, a nitrogen source, a magnesium salt, Tween 80 and water;

[0071] Based on the total volume of the fermentation medium, corn flour 10-30 g / L, glucose 20-45 g / L, soluble starch 5-15 g / L, yeast extract powder 10-15 g / L, peptone 15-20 g / L, magnesium sulfate 0.2-1 g / L, Tween 80 0.5-2 g / L, complex amino acid 2-4 g / L, the balance is deionized water, and the initial osmotic pressure of the fermentation medium at 0.05 MPa is 400-440 mOsm / kg;

[0072] (2) Preparation of seed solution: The Clostridium butyricum glycerol strain was inoculated into the seed culture medium at an inoculation rate of 1-3‰ (v / v) for seed culture, and the whole process was performed aseptically. The culture was carried out in a constant temperature incubator at 36-38°C for 14-20 hours to obtain a mature seed solution;

[0073] (3) Fermentation culture: The prepared fermentation medium was added into the fermentation tank at a volume of 70-80% (v / v) and sterilized; the prepared seed liquid was inoculated at a volume of 5×10 8 -1.5×10 9 CFU / L were inoculated into the fermentation tank and fermented at a temperature of 37±1℃ and a pH of 6-7;

[0074] After 12-14 hours of fermentation, 1.45-4.6 g / L of an inorganic ion source (the inorganic ion source is added in the form of a solution, and the inorganic ion source includes CaCl2, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and manganese sulfate) is added to the fermenter, and the culture is continued for 4-6 hours to terminate the fermentation;

[0075] (4) Fermentation broth induction culture: After the fermentation culture is completed, the tank is placed and 5-15v% of the fermentation medium volume of Lactobacillus plantarum fermentation broth (the strain preservation number of Lactobacillus plantarum is CGMCC No.15013, and the addition amount of Lactobacillus plantarum is 2×10 11 -7.5×10 11 CFU / L), and continue fermentation culture.

[0076] The present invention will be described in detail below through examples.

[0077] In the following examples, the specification of the anaerobic bottle is 100 mL, and the specification of the fermentation tank is 5 L.

[0078] The osmotic pressure of fermentation culture was adopted from Germany The company produces the model -OM806m freezing point osmometer for measurement.

[0079] Preparation method of Lactobacillus plantarum fermentation broth: 3×10 9 -9×10 9The inoculation amount of CFU / L is inoculated into MRS culture medium, and cultured at 37°C for 16-20 hours to obtain Lactobacillus plantarum fermentation liquid.

[0080] The strain deposit number of Lactobacillus plantarum is CGMCC No.15013 (disclosed in CN109423467A).

[0081] The source of Clostridium butyricum is COFCO Biotech Co., Ltd., and the strain deposit number is CGMCC No. 20417 (disclosed in CN112980735A).

[0082] The number of beneficial bacteria of Clostridium butyricum in the fermentation broth was determined using the method of "NY / T 4347-2023 Feed Additive Clostridium butyricum".

[0083] Spore rate in fermentation broth (%) = 100% × (spore number / viable bacteria number), where the spore number is the viable bacteria counted by dilution and coating on a plate after 10 minutes of 80°C water bathing.

[0084] The contents of acetic acid and butyric acid in the fermentation broth were detected using an Agilent 6890N gas chromatograph (equipped with a FID detector).

[0085] Unless otherwise specified, materials were commercially available.

[0086] Example 1

[0087] (1) Preparation of culture medium:

[0088] The seed culture medium includes: peptone 10g / L, beef powder 10g / L, yeast powder 3g / L, glucose 5g / L, soluble starch 1g / L, sodium chloride 5g / L, ammonium acetate 3g / L, L-cysteine ​​salt 0.5g / L, agar 0.5g / L, and the balance is deionized water;

[0089] The fermentation medium includes: 10 g / L corn flour, 20 g / L glucose, 5 g / L soluble starch, 10 g / L yeast extract powder, 15 g / L peptone, 0.2 g / L magnesium sulfate, 0.5 g / L Tween 80, 2 g / L composite amino acid (the mass ratio of lysine, methionine and glutamic acid in the composite amino acid is 1:0.5:0.5), and the balance is deionized water. The initial osmotic pressure of the fermentation medium at 0.05 MPa is 400 mOsm / kg.

[0090] The prepared seed culture medium and fermentation medium were adjusted to an initial pH of 6.8 ± 0.2 and sterilized at 121 °C for 20 min;

[0091] (2) Preparation of seed solution: The Clostridium butyricum glycerol strain was inoculated at a rate of 1‰ (v / v) into an anaerobic bottle containing a seed culture medium with a liquid volume of 40% (v / v) for activation culture (i.e., 40 μL of Clostridium butyricum glycerol strain was inoculated into a 100 mL anaerobic bottle containing 40 mL of seed culture medium). The whole process was performed aseptically and cultured in a constant temperature incubator at 36°C for 14 h to obtain a mature seed solution;

[0092] (3) Fermentation culture: The prepared fermentation medium was loaded into the fermentation tank at 70% (v / v) of the liquid volume (i.e., a 5 L fermentation tank was loaded with 3.5 L of fermentation medium), and sterilized at 121°C for 20 min; the prepared seed liquid was inoculated at a volume of 5 × 10 8 CFU / L was inoculated into the fermenter, and 0.1 vvm of sterile nitrogen was initially introduced to maintain the pressure at 0.05 MPa, the stirring speed at 100 rpm, the temperature at 37 ± 0.3 °C, and the pH at 6.7;

[0093] After 12 hours of fermentation, the stirring speed was controlled to 100 rpm, the temperature was 37±0.3°C, the pH was maintained at 6.7, and 2.5 g / L of inorganic ion source solution (the inorganic ion source solution included 0.2 g / L CaCl2, 0.6 g / L dipotassium hydrogen phosphate, 0.6 g / L potassium dihydrogen phosphate and 0.05 g / L manganese sulfate) was added to the fermenter, and the culture was continued for 4 hours to terminate the fermentation;

[0094] At the end of fermentation, the number of viable Clostridium butyricum in the fermentation broth was 2.7×10 9 CFU / mL, spore rate was 94%;

[0095] (4) Induction culture of fermentation broth: After the fermentation culture is completed, the tank is placed, 10 g / L of dipotassium hydrogen phosphate and 10 g / L of potassium dihydrogen phosphate are added to the fermentation broth, and after stirring evenly and leaving for 16 hours, the spore rate in the fermentation broth is measured to be 100%.

[0096] Example 2

[0097] (1) Preparation of culture medium:

[0098] The seed culture medium includes: peptone 10g / L, beef powder 10g / L, yeast powder 3g / L, glucose 5g / L, soluble starch 1g / L, sodium chloride 5g / L, ammonium acetate 3g / L, L-cysteine ​​salt 0.5g / L, agar 0.5g / L, and the balance is deionized water;

[0099] The fermentation medium includes: 30 g / L corn flour, 45 g / L glucose, 15 g / L soluble starch, 15 g / L yeast extract powder, 20 g / L peptone, 1 g / L magnesium sulfate, 2 g / L Tween 80, 4 g / L composite amino acid (the mass ratio of lysine, methionine and glutamic acid in the composite amino acid is 1:1:1), and the balance is deionized water. The initial osmotic pressure of the fermentation medium at 0.05 MPa is 440 mOsm / kg.

[0100] The prepared seed culture medium and fermentation medium were adjusted to an initial pH of 6.8 ± 0.2 and sterilized at 121 °C for 20 min;

[0101] (2) Preparation of seed solution: The Clostridium butyricum glycerol strain was inoculated at a rate of 1‰ (v / v) into an anaerobic bottle containing a seed culture medium with a liquid volume of 40% (v / v) for activation culture (i.e., 40 μL of Clostridium butyricum glycerol strain was inoculated into a 100 mL anaerobic bottle containing 40 mL of seed culture medium). The whole process was performed aseptically and cultured in a constant temperature incubator at 36°C for 14 h to obtain a mature seed solution;

[0102] (3) Fermentation culture: The prepared fermentation medium was loaded into the fermentation tank at 70% (v / v) of the liquid volume (i.e., a 5 L fermentation tank was loaded with 3.5 L of fermentation medium), and sterilized at 121°C for 20 min; the prepared seed liquid was inoculated at an inoculum of 1.5 × 10 9 CFU / L were inoculated into the fermenter, and 0.1 vvm of sterile nitrogen was initially introduced to maintain the pressure at 0.05 MPa, the stirring speed at 100 rpm, the temperature at 37 ± 0.3 °C, and the pH at 6.5;

[0103] After 12 hours of fermentation, the stirring speed was controlled to 100 rpm, the temperature was 37±0.3°C, the pH was maintained at 6.3, 4.6 g / L of inorganic ion source was added to the fermenter (the inorganic ion source was added in the form of a solution, including 0.4 g / L CaCl2, 2 g / L dipotassium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate and 0.2 g / L manganese sulfate), and the culture was continued for 6 hours to terminate the fermentation;

[0104] At the end of fermentation, the number of viable Clostridium butyricum in the fermentation broth was 3×10 9 CFU / mL, spore rate was 95%;

[0105] (4) Induction culture of fermentation broth: After the fermentation culture is completed, the tank is opened and 14 g / L CaCl2 is added to the fermentation broth. After stirring evenly and leaving for 12 hours, the spore rate in the fermentation broth is measured to be 100%.

[0106] Example 3

[0107] (1) Preparation of culture medium:

[0108] The seed culture medium includes: peptone 10g / L, beef powder 10g / L, yeast powder 3g / L, glucose 5g / L, soluble starch 1g / L, sodium chloride 5g / L, ammonium acetate 3g / L, L-cysteine ​​salt 0.5g / L, agar 0.5g / L, and the balance is deionized water;

[0109] The fermentation medium includes: 22 g / L corn flour, 30 g / L glucose, 10 g / L soluble starch, 15 g / L yeast extract powder, 15 g / L peptone, 0.8 g / L magnesium sulfate, 1 g / L Tween 80, 4 g / L composite amino acid (the mass ratio of lysine, methionine and glutamic acid in the composite amino acid is 1:1:1), and the balance is deionized water. The initial osmotic pressure of the fermentation medium at 0.05 MPa is 415 mOsm / kg.

[0110] The prepared seed culture medium and fermentation medium were adjusted to an initial pH of 6.8 ± 0.2 and sterilized at 121 °C for 20 min;

[0111] (2) Preparation of seed solution: The Clostridium butyricum glycerol strain was inoculated at a rate of 1‰ (v / v) into an anaerobic bottle containing a seed culture medium with a liquid volume of 40% (v / v) for activation culture (i.e., 40 μL of Clostridium butyricum glycerol strain was inoculated into a 100 mL anaerobic bottle containing 40 mL of seed culture medium). The whole process was performed aseptically and cultured in a constant temperature incubator at 36°C for 14 h to obtain a mature seed solution;

[0112] (3) Fermentation culture: The prepared fermentation medium was loaded into the fermentation tank at 70% (v / v) of the liquid volume (i.e., a 5-L fermentation tank was loaded with 3.5 L of fermentation medium), and sterilized at 121°C for 20 min; the prepared seed liquid was inoculated at an inoculum of 1×10 9 CFU / L was inoculated into the fermenter, and 0.1 vvm of sterile nitrogen was initially introduced to maintain the pressure at 0.05 MPa, the stirring speed at 100 rpm, the temperature at 37 ± 0.3 °C, and the pH at 6.5;

[0113] After 12 hours of fermentation, the stirring speed was controlled to 100 rpm, the temperature was 37±0.3°C, the pH was maintained at 6.3, 2.8 g / L of inorganic ion source was added to the fermenter (the inorganic ion source was added in the form of a solution, including 0.3 g / L of CaCl2, 1.2 g / L of dipotassium hydrogen phosphate, 1.2 g / L of potassium dihydrogen phosphate and 0.1 g / L of manganese sulfate), and the culture was continued for 6 hours to terminate the fermentation;

[0114] At the end of fermentation, the number of viable Clostridium butyricum in the fermentation broth was 3.2×10 9 CFU / mL, spore rate was 96%;

[0115] (4) Fermentation broth induction culture: After the fermentation culture was completed, the tank was placed and 10% by volume of the fermentation medium of Lactobacillus plantarum fermentation broth (the strain preservation number of Lactobacillus plantarum is CGMCC No. 15013, and the addition amount of Lactobacillus plantarum is 4.5×10 11 CFU / L), and after being stirred evenly and placed for 20 hours, the spore rate in the fermentation liquid was measured to be 100%.

[0116] Example 4

[0117] (1) Preparation of culture medium:

[0118] The seed culture medium includes: peptone 10g / L, beef powder 10g / L, yeast powder 3g / L, glucose 5g / L, soluble starch 1g / L, sodium chloride 5g / L, ammonium acetate 3g / L, L-cysteine ​​salt 0.5g / L, agar 0.5g / L, and the balance is deionized water;

[0119] The fermentation medium includes: 16 g / L corn flour, 34 g / L glucose, 12 g / L soluble starch, 15 g / L yeast extract powder, 15 g / L peptone, 0.8 g / L magnesium sulfate, 1.5 g / L Tween 80, 3.6 g / L composite amino acid (the mass ratio of lysine, methionine and glutamic acid in the composite amino acid is 1:0.8:0.8), and the balance is deionized water. The initial osmotic pressure of the fermentation medium at 0.05 MPa is 425 mOsm / kg.

[0120] The prepared seed culture medium and fermentation medium were adjusted to an initial pH of 6.8 ± 0.2 and sterilized at 121 °C for 20 min;

[0121] (2) Preparation of seed solution: The Clostridium butyricum glycerol strain was inoculated at a rate of 1‰ (v / v) into an anaerobic bottle containing a seed culture medium with a liquid volume of 40% (v / v) for activation culture (i.e., 40 μL of Clostridium butyricum glycerol strain was inoculated into a 100 mL anaerobic bottle containing 40 mL of seed culture medium). The whole process was performed aseptically and cultured in a constant temperature incubator at 36°C for 14 h to obtain a mature seed solution;

[0122] (3) Fermentation culture: The prepared fermentation medium was loaded into the fermentation tank at 70% (v / v) of the liquid volume (i.e., a 5-L fermentation tank was loaded with 3.5 L of fermentation medium), and sterilized at 121°C for 20 min; the prepared seed liquid was inoculated at an inoculum of 1.2 × 10 9 CFU / L was inoculated into the fermenter, and 0.1 vvm of sterile nitrogen was initially introduced to maintain the pressure at 0.05 MPa, the stirring speed at 100 rpm, the temperature at 37 ± 0.3 °C, and the pH at 6.5;

[0123] After 12 hours of fermentation, the stirring speed was controlled to 100 rpm, the temperature was 37±0.3°C, the pH was maintained at 6.5, 3.2 g / L of inorganic ion source was added to the fermenter (the inorganic ion source was added in the form of a solution, including 0.25 g / L of CaCl2, 1.4 g / L of dipotassium hydrogen phosphate, 1.4 g / L of potassium dihydrogen phosphate and 0.15 g / L of manganese sulfate), and the culture was continued for 6 hours to terminate the fermentation;

[0124] At the end of fermentation, the number of viable Clostridium butyricum in the fermentation broth was 3.2×10 9 CFU / mL, spore rate was 97%;

[0125] (4) Fermentation broth induction culture: After the fermentation culture was completed, the tank was placed and 5% by volume of the fermentation medium of Lactobacillus plantarum fermentation broth (the strain preservation number of Lactobacillus plantarum is CGMCC No. 15013, and the addition amount of Lactobacillus plantarum is 2×10 11 CFU / L), and after being stirred evenly and placed for 20 hours, the spore rate in the fermentation liquid was measured to be 100%.

[0126] Example 5

[0127] The method of Example 3 was followed, except that in step (3), the amount of inorganic ion source added was 5.8 g / L, and the number of viable Clostridium butyricum in the fermentation broth was measured at the end of fermentation to be 2.2×10 9 CFU / mL, the spore rate was 88%; after induction culture of the fermentation broth, the spore rate in the fermentation broth was measured to be 92%.

[0128] Example 6

[0129] The method of Example 3 was followed, except that in step (3), after 10 h of fermentation, an inorganic ion source was added to the fermenter and the culture was continued for 8 h (the total fermentation time was the same as that of Example 3). At the end of the fermentation, the number of viable Clostridium butyricum in the fermentation broth was measured to be 2.4×10 9 CFU / mL, the spore rate was 95%; after induction culture of the fermentation broth, the spore rate in the fermentation broth was measured to be 100%.

[0130] Example 7

[0131] The method of Example 3 is followed, except that in step (4), the Lactobacillus plantarum fermentation broth is replaced with Bacillus subtilis fermentation broth, and after being stirred evenly and allowed to stand for 20 hours, the spore rate in the fermentation broth is measured to be 95%.

[0132] Example 8

[0133] The method of Example 3 was followed, except that in step (4), CaCO3 was added to the fermentation broth, and after stirring evenly and standing for 12 hours, the spore rate in the fermentation broth was measured to be 95%.

[0134] Example 9

[0135] The method of Example 3 was followed, except that in step (4), MnSO4 was added to the fermentation broth, and after stirring evenly and standing for 12 hours, the spore rate in the fermentation broth was measured to be 95%.

[0136] Comparative Example 1

[0137] The method of Example 3 is different in that the composition of the fermentation medium is different, as follows:

[0138] The fermentation medium includes: 30 g / L corn flour, 45 g / L glucose, 15 g / L soluble starch, 15 g / L yeast extract powder, 20 g / L peptone, 1 g / L magnesium sulfate, 1 g / L Tween 80, 4 g / L composite amino acid (the mass ratio of lysine, methionine and glutamic acid in the composite amino acid is 1:1:1), 0.4 g / L CaCl2, 2 g / L dipotassium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 0.2 g / L manganese sulfate, and the balance is deionized water. The initial osmotic pressure of the fermentation medium at 0.05 MPa is 670 mOsm / kg.

[0139] The fermentation was terminated after 22 h of culture, and the number of viable Clostridium butyricum in the fermentation broth was measured to be 1.58×10 9 CFU / mL, and the spore rate was 68%.

[0140] Comparative Example 2

[0141] The method of Example 3 was followed, except that in step (3), the inorganic ion source was replaced with sodium acetate, and the fermentation was terminated after 22 h of fermentation. The viable count of Clostridium butyricum in the fermentation broth was measured to be 2.38×10 9 CFU / mL, and the spore rate was 70%.

[0142] Comparative Example 3

[0143] Comparative Example 3-1

[0144] The method of Example 3 was followed, except that in step (3), 20 g / L of glucose and 10 g / L of yeast powder were added to the fermentation tank, and the fermentation was terminated after 22 h of fermentation. The viable count of Clostridium butyricum in the fermentation broth was measured to be 1.77 × 10 9 CFU / mL, and the spore rate was 62%.

[0145] Comparative Example 3-2

[0146] The method of Comparative Example 3-1 was followed except that the fermentation time was prolonged, i.e., the number of viable Clostridium butyricum in the fermentation broth was measured to be 1.5×109 CFU / mL, and the spore rate was 65%.

[0147] Comparative Example 4

[0148] The method of Example 3 is different in that in step (4), no Lactobacillus plantarum fermentation broth is added to the fermentation broth, and the culture is continued for 20 hours. The spore rate in the fermentation broth is measured to be 86%.

[0149] Test Example 1

[0150] The dynamic changes of the live count of Clostridium butyricum in the fermentation broth of the above examples and comparative examples were monitored throughout the fermentation cycle.

[0151] The monitoring method includes taking samples every 2 hours to determine the number of live bacteria of Clostridium butyricum in the fermentation broth of the embodiment and the comparative example, and drawing a curve of the change of the number of live bacteria of Clostridium butyricum in the fermentation broth with the fermentation time, and calculating the fermentation intensity of Clostridium butyricum. The results are shown in Tables 1 and Figure 1 .

[0152] Fermentation intensity (10 8 CFU / (mL . h)) = 100% × (the number of viable Clostridium butyricum in the fermentation broth after the fermentation in step (3) - the number of viable Clostridium butyricum in the fermentation medium at 0 h of fermentation) / fermentation period.

[0153] Test Example 2

[0154] The dynamic changes of the spore rate of Clostridium butyricum in the fermentation broth were monitored throughout the fermentation cycle of the above examples and comparative examples.

[0155] The monitoring method includes taking samples every 2 hours to measure the spore rate of Clostridium butyricum in the fermentation broth of the embodiment and the comparative example, and drawing a curve chart of the change of the spore rate of Clostridium butyricum in the fermentation broth with the fermentation time. The results are shown in Table 2 and Figure 2 .

[0156] Test Example 3

[0157] The contents of acetic acid and butyric acid in the fermentation broth after induction culture of the above examples and comparative examples were respectively measured, and the results are shown in Table 3.

[0158] Test Example 4

[0159] The heat resistance and gastric acid and bile resistance of the spores in the fermentation broth after induction culture of the above examples and comparative examples were tested, and the results are shown in Table 3.

[0160] The heat resistance test method of spores includes: taking 1 mL of the fermentation liquid after induction culture and heating it in a water bath at 80°C for 10 minutes, and calculating the survival rate of the strains, where the survival rate (%) = (the number of live bacteria after heat treatment / the number of live bacteria before treatment) × 100%.

[0161] The gastric acid resistance test method of spores includes: taking 10 mL of prepared artificial gastric juice, filtering through a 0.22 μm filter membrane, adding 1 mL of fermentation broth after induction culture, mixing, culturing at 37° C., taking samples at 0 h, 2 h and 4 h, respectively, and after dilution, using a plate colony counting method to analyze the strain's tolerance to artificial gastric juice, and calculating the survival rate of the strain, the survival rate (%) = (the number of live bacteria after treatment / the number of live bacteria before treatment) × 100%.

[0162] The bile salt tolerance test method of spores was as follows: 1 mL of fermentation broth was added to 10 mL of 0.3% bile salt solution, mixed, cultured at 37°C, and samples were taken at 0 h, 2 h, and 4 h, respectively. After dilution, the tolerance of the strain to bile salts was analyzed by the plate colony counting method, and the survival rate of the strain was calculated. Survival rate (%) = (number of live bacteria after treatment / number of live bacteria before treatment) × 100%.

[0163] Table 1

[0164]

[0165] Table 2

[0166]

[0167]

[0168] Table 3

[0169]

[0170] By adopting the method of the present invention, the initial osmotic pressure of the fermentation medium is optimized, an inorganic ion source is supplemented at the end of the logarithmic growth period, and a protective agent is added after the fermentation tank is stopped, thereby effectively promoting the proliferation and spore production of Clostridium butyricum, achieving high-intensity fermentation and high spore conversion rate, and shortening the fermentation cycle; at the same time, after the fermentation is completed, the spore rate can be further increased to 100%, fully mature spores are obtained, and the heat resistance and gastric acid and bile salt resistance of the spores are improved; in addition, the content of short-chain fatty acids (especially acetic acid and butyric acid) in the fermentation liquid is also greatly increased.

[0171] As can be seen from Examples 1-4, in the preferred embodiment, the fermentation period of Clostridium butyricum is 16-18 h, and the fermentation intensity reaches 1.775×10 8 CFU / mL . h, and the number of viable Clostridium butyricum in the fermentation broth reached 3.2×10 9 CFU / mL, and the spore rate reached 97%; after the induction culture, the spore rate further increased to 100%. The spores have excellent heat resistance and gastric acid and bile salt resistance, and the content of acetic acid in the fermentation broth is as high as 12.32g / L, and the content of butyric acid is as high as 34.16g / L.

[0172] By comparing Example 3 with Examples 5-9, it can be seen that the preferred Clostridium butyricum fermentation culture method can further improve the fermentation intensity and spore transformation of Clostridium butyricum, shorten the fermentation cycle, and further improve the heat resistance and gastric acid and bile resistance of the spores, and promote the synthesis of its fermentation metabolites acetic acid and butyric acid.

[0173] Comparative Example 1 uses a medium with a high inorganic salt content and a high initial osmotic pressure, which results in a decrease in the fermentation intensity and spore conversion rate of Clostridium butyricum, and a prolonged fermentation cycle; the butyric acid content in the fermentation broth is reduced, and the heat resistance and gastric acid bile resistance of the spores are also deteriorated; Comparative Example 2 changes the type of supplemented inorganic ion source, which results in a decrease in the fermentation intensity and spore conversion rate of Clostridium butyricum, and a prolonged fermentation cycle; the butyric acid content in the fermentation broth is reduced, and the heat resistance and gastric acid bile resistance of the spores are also deteriorated; Comparative Example 3-1 adds 2-4-nitropropene to the fermenter at the end of the fermentation culture. Adding carbon and nitrogen sources (glucose and yeast powder) resulted in reduced fermentation intensity and spore conversion rate of Clostridium butyricum, and prolonged fermentation cycle; Comparative Example 3-2 extended the fermentation time on the basis of Comparative Example 3-1, but the spore rate still could not be increased to 100%, the butyric acid content in the fermentation broth was reduced, and the heat resistance and gastric acid and bile resistance of the spores also deteriorated; Comparative Example 4 did not add a protective agent during the induction culture stage, and continued fermentation culture, but the spore rate still could not be increased to 100%, the acetic acid and butyric acid contents in the fermentation broth were reduced, and the heat resistance and gastric acid and bile resistance of the spores also deteriorated.

[0174] In summary, the method of the embodiment of the present invention can improve the fermentation intensity and spore conversion rate of Clostridium butyricum, shorten the fermentation cycle; further improve spore conversion, obtain fully mature spores, and improve spore performance; at the same time, greatly increase the content of short-chain fatty acids (especially acetic acid and butyric acid) in the fermentation broth.

[0175] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for improving the fermentation intensity and spore transformation of Clostridium butyricum, characterized in that: The method comprises: inoculating Clostridium butyricum into a fermentation medium for fermentation culture; adding an inorganic ion source to the fermentation liquid at the end of the logarithmic growth period of the fermentation culture to continue the fermentation culture until the spore rate is not less than 90%; then, optionally, adding a protective agent to the fermentation liquid for induction culture to further increase the spore rate; Wherein, the initial osmotic pressure of the fermentation medium at 0.05 MPa is ≤460 mOsm / kg; Wherein, the inorganic ion source is selected from at least one of a potassium ion source, a manganese ion source and a calcium ion source.

2. The method according to claim 1, wherein: The initial osmotic pressure of the fermentation medium at 0.05 MPa is 400-440 mOsm / kg; And / or, the inorganic ion source is added 12-16 hours after inoculation, preferably 12-14 hours; And / or, based on the total volume of the fermentation medium, the inoculation amount of Clostridium butyricum is 5×10 8 -1.5×10 9 CFU / L.

3. The method according to claim 1, wherein: The protective agent is selected from at least one of potassium salt, calcium salt and Lactobacillus plantarum; Preferably, the potassium salt is selected from potassium dihydrogen phosphate and / or dipotassium hydrogen phosphate; Preferably, the strain accession number of the Lactobacillus plantarum is CGMCC No.15013; More preferably, the plant lactobacillus is provided in the form of a plant lactobacillus fermentation broth, and the preparation method of the plant lactobacillus fermentation broth comprises: 9 -9×10 9 The inoculation amount of CFU / L is inoculated into MRS culture medium, and cultured at 37±1°C for 16-20h to obtain Lactobacillus plantarum fermentation liquid.

4. The method according to claim 1 or 2, wherein: Based on the total volume of the fermentation medium, the addition amount of the inorganic ion source is 1.45-4.6 g / L; Preferably, the potassium ion source is selected from potassium dihydrogen phosphate and / or dipotassium hydrogen phosphate; Preferably, the manganese ion source is selected from manganese sulfate and / or manganese chloride; Preferably, the calcium ion source is selected from calcium chloride and / or calcium carbonate.

5. The method according to claim 3, wherein: The protective agent is potassium salt, and the amount of the protective agent added is 2-25 g / L, preferably 5-12 g / L, based on the total volume of the fermentation medium; Alternatively, the protective agent is a calcium salt, and the amount of the protective agent added is 2-20 g / L, preferably 8-14 g / L, based on the total volume of the fermentation medium; Alternatively, the protective agent is Lactobacillus plantarum, and the amount of the protective agent added is 1×10 11 -10×10 11 CFU / L, preferably 2×10 11 -7.5×10 11 CFU / L.

6. The method according to any one of claims 1 to 5, wherein: The fermentation medium comprises a carbon source, a nitrogen source, a magnesium salt, Tween 80 and water; Preferably, the fermentation medium comprises corn flour, glucose, soluble starch, yeast extract powder, peptone, magnesium sulfate, Tween 80, amino acids and water; More preferably, the amino acids include lysine, glutamic acid and methionine; More preferably, the mass ratio of lysine, glutamic acid and methionine in the amino acids is 1:(0.5-1):(0.5-1).

7. The method according to claim 6, wherein: Based on the total volume of the fermentation medium, the content of the corn flour is 10-30 g / L, the content of the glucose is 20-45 g / L, the content of the soluble starch is 5-15 g / L, the content of the yeast extract is 10-15 g / L, the content of the peptone is 15-20 g / L, the content of the magnesium sulfate is 0.2-1 g / L, the content of the Tween 80 is 0.5-2 g / L, the content of the amino acid is 2-4 g / L, and the balance is water.

8. The method according to any one of claims 1 to 5, wherein: The method further comprises: before inoculating into the fermentation medium, inoculating the Clostridium butyricum glycerol bacteria into the seed culture medium for seed culture to obtain a seed solution containing Clostridium butyricum; Preferably, the seed culture medium is RCM culture medium; Preferably, the inoculation amount of the Clostridium butyricum glycerol bacteria is 1-3v‰ of the volume of the seed culture medium; And / or, the strain collection number of the Clostridium butyricum is CGMCC No.20417.

9. The method according to claim 8, wherein: The seed culture conditions include: temperature of 36-38° C., time of 14-20 h, pH of 5.2-7, and anaerobic culture.

10. The method according to any one of claims 1 to 5, wherein: The fermentation culture conditions include: temperature of 36-38°C, pH of 6-7, and anaerobic culture; And / or, the induction culture conditions include: temperature of 36-38°C and time of 12-24h.

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