A composite probiotic system with high butyric acid production and preparation method thereof

Abstract

The invention provides a butyrate probiotic culture medium, comprising the following components: 6-12 g / L of peptone, 6-12 g / L of beef extract, 1-5 g / L of yeast extract, 4-8 g / L of glucose, 4-8 g / L of sodium chloride, 0.5-2 g / L of soluble starch, 1-5 g / L of sodium acetate, 0.1-1.0 g / L of L-cysteine hydrochloride, 4-8 g / L of xylan, 1-3 g / L of dipotassium hydrogen phosphate, 0.1-1.0 g / L of magnesium sulfate heptahydrate, and 0.01-0.05 g / L of ferrous sulfate. The invention also specifically provides a method for culturing a high-butyrate cooperative system of Lactobacillus plantarum and Clostridium tyrobutyricum using the culture medium. The method not only improves the butyrate yield but also reduces the production cost of butyrate bioconversion, thereby having significant economic benefits.

Description

Technical Field

[0001] The invention belongs to microbial technology and relates to a composite probiotic system with high butyrate production and a preparation method thereof. Background Art

[0002] Butyrate is a typical short-chain fatty acid (SCFA) that has attracted widespread attention due to its important role in maintaining body health, such as providing energy for colonocytes, regulating intestinal homeostasis, and modulating intestinal inflammation. Numerous studies have found that most patients with chronic non-communicable diseases have lower intestinal butyrate levels than healthy individuals. Some studies even suggest that intestinal butyrate levels can serve as a potential biomarker for assessing the health of an organism. Therefore, butyrate is an important physiologically active substance, and the development of safe, low-cost, and efficient methods for its production is of great significance.

[0003] Currently, butyric acid is primarily derived from chemical synthesis and microbial fermentation, with the majority of commercially available butyric acid derived primarily from chemical synthesis. Common chemical synthesis methods for butyric acid production include propylene carbonylation and n-butyraldehyde oxidation. However, chemical synthesis not only consumes fossil raw materials but also produces environmentally harmful byproducts, such as toxic waste metal catalysts (sulfides, cyanides, and chlorides).

[0004] Compared with chemical synthesis of butyric acid, the production of butyric acid through microbial conversion will be more promising in the future. This advantage is mainly reflected in two key aspects. First, the production of butyric acid by microbial fermentation can utilize low-cost, renewable biomass raw materials or agricultural processing waste as fermentation substrates, which not only reduces production costs but is also more environmentally friendly. Secondly, butyric acid produced by microbial fermentation has higher biosafety and is more suitable for application in the food and pharmaceutical industries. Although studies have shown that Clostridium tyrobutyricum is a butyric acid-producing bacterium with great development and application value, due to its limitations in fermentation substrates (carbon sources) and low butyric acid production, there is still a lack of research on the efficient utilization of the butyric acid metabolic capacity of Clostridium tyrobutyricum. Therefore, there is an urgent need for a method for preparing a probiotic that can efficiently produce butyric acid. Summary of the Invention

[0005] The present invention provides a butyrate probiotic culture medium, comprising the following components: 6-12 g / L of peptone, 6-12 g / L of beef extract, 1-5 g / L of yeast extract, 4-8 g / L of glucose, 4-8 g / L of sodium chloride, 0.5-2 g / L of soluble starch, 1-5 g / L of sodium acetate, 0.1-1.0 g / L of L-cysteine hydrochloride, 4-8 g / L of xylan, 1-3 g / L of dipotassium hydrogen phosphate, 0.1-1.0 g / L of magnesium sulfate heptahydrate, and 0.01-0.05 g / L of ferrous sulfate;

[0006] Furthermore, the butyrate probiotic culture medium comprises the following components: 6-12 g / L peptone, 6-12 g / L beef extract, 1-5 g / L yeast extract, 4-8 g / L glucose, 4-8 g / L sodium chloride, 0.5-2 g / L soluble starch, 1-5 g / L sodium acetate, 0.1-1.0 g / L L-cysteine hydrochloride, 4-8 g / L xylan, 1-3 g / L dipotassium hydrogen phosphate, 0.1-1.0 g / L magnesium sulfate heptahydrate, and 0.01-0.05 g / L ferrous sulfate.

[0007] A second aspect of the present invention provides a butyrate probiotics cultivation method comprising the following steps:

[0008] S1: taking Lactobacillus plantarum and Clostridium tyrobutyricum bacterial culture solutions respectively, mixing them with 40-60% v / v sterile glycerol and freezing them to obtain frozen solutions of the two strains, taking appropriate amounts of glycerol frozen solutions of Lactobacillus plantarum and Clostridium tyrobutyricum, respectively, adding them to 20-30 times the volume of MRS medium and RCM medium, respectively, and pre-culturing them under anaerobic conditions at 35-40° C., wherein Lactobacillus plantarum is cultured for 20-30 hours and Clostridium tyrobutyricum is cultured for 40-55 hours, to obtain pre-culture solutions of Lactobacillus plantarum and Clostridium tyrobutyricum;

[0009] S2: Take appropriate amounts of the pre-cultured solutions of Lactobacillus plantarum and Clostridium tyrobutyricum, add 8-10 times the volume of MRS medium and RCM medium, respectively, and culture anaerobically at 35-40°C, wherein Lactobacillus plantarum is cultured for 10-15 hours and Clostridium tyrobutyricum is cultured for 30-40 hours, and obtain the bacterial solution at the end of the logarithmic growth phase, wash, and add sterile water to obtain the OD 600 It is a bacterial suspension of 0.5 to 0.8;

[0010] S3: inoculating the bacterial suspension of Lactobacillus plantarum and Clostridium tyrobutyricum into any of the butyrate probiotic culture media, and fermenting for 4 to 65 hours; further, fermenting for 32 to 60 hours;

[0011] Furthermore, the plant lactobacillus is Lactobacillus plantarum C0502, taxonomically named Lactiplantibacillus plantarum, which has been deposited in the General Microbiology Center of China Culture Collection Administration, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 31485, and the deposit date is July 29, 2024;

[0012] Furthermore, the Clostridium tyrobutyricum is Clostridium tyrobutyricum ATCC25755;

[0013] Furthermore, in S2, the inoculation amount of the Lactobacillus plantarum is 0.5-2% v / v; furthermore, the inoculation amount of the Lactobacillus plantarum is 1% v / v;

[0014] Furthermore, in S2, the inoculation amount of Clostridium tyrobutyricum is 0.5-2% v / v; furthermore, the inoculation amount of Lactobacillus plantarum is 1% v / v; BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The effects of different culture methods on butyrate production.

[0016] Figure 2 Butyrate production by co-culture of Lactobacillus plantarum C0502 and Clostridium tyrobutyricum in RCM containing different chemical components.

[0017] Figure 3 The pH changes in RCM culture media with different culture methods and containing different chemical components;

[0018] Figure 4 The growth curves (OD 600 );

[0019] Figure 5 is the change of pH in the final culture medium under different culture modes;

[0020] Figure 6 is the change of glucose in the final culture medium under different culture modes;

[0021] Figure 7 is the change in acid concentration during the monoculture of Lactobacillus plantarum C0502 in the final medium;

[0022] Figure 8 is the change in acid concentration during the monoculture of Clostridium tyrobutyricum in the final medium;

[0023] Figure 9 It is the change of acid concentration during the co-culture of Lactobacillus plantarum C0502 and Clostridium tyrobutyricum in the final culture medium.

[0024] Beneficial effects

[0025] Through experiments, the present invention screened out probiotics Lactobacillus plantarum C0502 and Clostridium tyrobutyricum ATCC25755 that can produce high butyrate. Clostridium tyrobutyricum ATCC25755 has two main butyrate metabolism systems: glucose-butyrate and lactate / acetate-butyrate. Lactobacillus plantarum C0502 can be promoted to grow and metabolize lactate by cheap biomass feedstock (xylan).

[0026] The present invention has demonstrated through experiments that a culture medium for efficiently producing butyrate has been discovered. The invention fully utilizes the interaction effect of xylan and Lactobacillus plantarum C0502 to produce lactic acid and the efficient lactic acid / acetic acid-butyrate conversion system of Clostridium tyrobutyricum ATCC25755 to construct a high-butyrate-producing probiotic interaction system, which not only increases butyrate production but also reduces the production cost of butyrate bioconversion, thus having significant economic benefits. DETAILED DESCRIPTION

[0027] The technical solutions of the present invention are further described below in conjunction with the accompanying drawings and specific embodiments, but these embodiments shall not be used to interpret the limitations of the present invention; the experimental methods in the following embodiments are conventional methods unless otherwise specified, and the test materials used in the following embodiments are commercially available unless otherwise specified; after reading the description of the present invention, various equivalent changes, modifications and modifications made by technical personnel in this field shall fall within the scope defined by the claims of the present invention.

[0028] (I) Reagents: Xylan (Chinook Therapeutics, Vancouver, Canada); chromatography-grade methanol (Sigma-Aldrich, Milan, Italy); lactic acid, acetic acid, and butyric acid standards (Macklin, Shanghai, China); glucose standard (Yuanye, Shanghai, China). All other reagents were of analytical grade.

[0029] (2) Culture medium:

[0030] 1. MRS medium: 10 g / L peptone, 20 g / L glucose, 10 g / L beef extract, 5 g / L yeast extract, 1 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L sodium citrate, 0.2 g / L magnesium sulfate heptahydrate, and 0.054 g / L manganese sulfate pentahydrate, for the culture and enumeration of lactic acid bacteria.

[0031] 2. Reinforced Clostridial medium (RCM): 10 g / L peptone, 10 g / L beef extract, 3 g / L yeast extract, 5 g / L glucose, 5 g / L sodium chloride, 1 g / L soluble starch, 3 g / L sodium acetate, and 0.5 g / L L-cysteine hydrochloride, for the culture and enumeration of Clostridia.

[0032] 3. Clostridium growth medium (CGM): 5 g / L peptone, 5 g / L yeast extract, 60 g / L glucose, 3 g / L ammonium sulfate, 0.6 g / L magnesium sulfate heptahydrate, 0.03 g / L ferrous sulfate, and 40 g / L calcium carbonate.

[0033] (3) Strains:

[0034] 1. Lactobacillus plantarum C0502 is deposited in the General Microbiology Center of China Culture Collection of Microorganisms with the deposit number CGMCC No. 31485.

[0035] Clostridium tyrobutyricum ATCC25755 was purchased from Henan Industrial Microbiology Engineering Technology Research Center (www.bncc.com).

[0036] (IV) Implementation

[0037] Example 1: Detection methods of various indicators

[0038] 1.1 OD 600 and pH

[0039] The OD600 of the bacterial suspension was measured using a microplate reader (BioTek Epoch 2, Agilent, California, USA) and the pH value of the bacterial fermentation broth was measured using a pH meter (Mettler Toledo, Shanghai, China).

[0040] 1.2 Detection of lactic acid, acetic acid and butyric acid by high performance liquid chromatography (HPLC)

[0041] 1.2.1 Sample Preparation: Add 0.25 mL of 150 g / L K₄Fe(CN)₆ and 0.25 mL of 300 g / L ZnSO₄ to 1 mL of sample, mix thoroughly, and let stand for 30 min. Subsequently, centrifuge the mixture at 10,000 g for 5 min, and filter the supernatant through a 0.22 μm filter.

[0042] 1.2.2 Liquid phase conditions: Instrument: HPLC (Vanquish, Thermo Fisher Scientific, USA); Chromatographic column: C 18Column (250 mm × 4.6 mm, 5 μm, Thermo Fisher Scientific, USA); mobile phase: A: chromatography-grade methanol; B: 50 mmol / L K2HPO4 (pH adjusted to 2.8 with phosphoric acid); gradient elution conditions: starting with a ratio of potassium dihydrogen phosphate:methanol = 95:5, isocratic elution for 7 min, linearly increasing the methanol concentration from 7 to 16 min to reach a ratio of potassium dihydrogen phosphate:methanol = 60:40 at 16 min, followed by isocratic elution with potassium dihydrogen phosphate:methanol = 60:40; all ratios are by volume; injection volume: 10 μL; flow rate: 1.0 mL / min; column temperature: 40°C; detector: UV detector (Thermo Fisher Scientific); detection wavelength: 210 nm.

[0043] 1.3 High Performance Liquid Chromatography (HPLC) Detection of Glucose

[0044] 1.3.1 Sample treatment: 1 mL of sample was centrifuged at 10,000 g for 5 minutes, and the supernatant was filtered through a 0.22 μm filter membrane.

[0045] 13.2 Liquid phase conditions: Instrument: HPLC (Agilent 1260, Agilent Technologies, USA); Chromatographic column: Aminex HPX-87H (300 mm × 7.8 mm, 5 μm, Shanghai, China); Mobile phase: 5 mmol / L sulfuric acid; Injection volume: 20 μL; Flow rate: 0.6 mL / min; Column temperature: 60°C; Detector: Refractive index detector (RID, 1260 Series, Agilent Technologies, USA).

[0046] Example 2: Optimization of co-fermentation medium

[0047] 2.1 Preservation of strains

[0048] 0.5 mL of bacterial culture solution (Lactobacillus plantarum C0502 or Clostridium tyrobutyricum ATCC25755) was mixed with 0.5 mL of 50% v / v sterile glycerol and then frozen in a -80°C refrigerator. These frozen solutions were used as seed solutions for subsequent activation culture.

[0049] 2.2 Activation of strains

[0050] 2.2.1 Activation of Lactobacillus plantarum

[0051] Take 0.2 mL of glycerol frozen solution of Lactobacillus plantarum C0502 and add it to 4.5 mL of MRS medium. Pre-culture it under anaerobic conditions at 37 ° C for 24 h. Then take 0.5 mL of pre-cultured bacterial solution and add it to 4.5 mL of MRS medium. Culture it under anaerobic conditions at 37 ° C for 12 h to obtain the bacterial solution at the end of the logarithmic growth phase. After washing twice, add sterile water and obtain the OD 600 =0.6 bacterial suspension. These bacterial suspensions serve as seed liquid for subsequent fermentation.

[0052] 2.2.2 Activation of Clostridium tyrobutyricum

[0053] Take 0.2 mL of glycerol frozen stock of Clostridium tyrobutyricum ATCC25755 and add it to 4.5 mL RCM medium. Incubate it under anaerobic conditions at 37 °C for 48 h. Then take 0.5 mL of pre-cultured bacterial solution and add it to 4.5 mL RCM medium. Incubate it under anaerobic conditions at 37 °C for 36 h. Get bacterial solution at the end of logarithmic growth phase. Wash it twice and add sterile water to get OD 600 =0.6 bacterial suspension. These bacterial suspensions serve as seed liquid for subsequent fermentation.

[0054] 2.3 Optimization of co-fermentation medium

[0055] The culture medium includes (a) RCM; (b) RCM + 5 g / 100 mL xylan; (c) RCM + 5 g / 100 mL xylan + 2 g / L potassium dihydrogen phosphate; (d) RCM + 5 g / 100 mL xylan + 2 g / L sodium citrate; (e) RCM + 5 g / 100 mL xylan + 0.6 g / L magnesium sulfate heptahydrate; (f) RCM + 5 g / 100 mL xylan + 0.054 g / L manganese sulfate pentahydrate; (g) RCM + 5 g / 100 mL xylan + 3 g / L ammonium sulfate; (h) RCM + 5 g / 100 mL xylan + 0.03 g / L ferrous sulfate; (i) RCM + 5 g / 100 mL xylan + 40 g / L calcium carbonate.

[0056] Each modified culture medium was inoculated with 1% v / v Lactobacillus plantarum, 1% v / v Clostridium tyrobutyricum, or a combination of 0.5% v / v Lactobacillus plantarum and 0.5% v / v Clostridium tyrobutyricum.

[0057] During the fermentation process, samples were collected at 12h, 24h, 48h and 72h to measure pH and butyrate concentration. Figure 1 、 Figure 2 and Figure 3As shown in the results, the butyrate production of the co-culture system of Lactobacillus plantarum C0502 and Clostridium tyrobutyricum was 2.35 times higher than that of the single culture system of Clostridium tyrobutyricum, increasing from 1.09 g / L to 2.57 g / L. Adding 5 g / 100 mL of xylan to RCM medium significantly increased the butyrate production of the co-culture system by 1.45 times, from 2.57 g / L to 3.75 g / L. Figure 1 Among them, the addition of potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and ferrous sulfate to the RCM medium containing xylan also increased the butyrate production in the co-culture system of Lactobacillus plantarum C0502 and Clostridium tyrobutyricum compared to the culture in the RCM medium containing xylan ( Figure 2 ).

[0058] Therefore, the components that effectively stimulate butyrate production in the co-culture system of Lactobacillus plantarum C0502 and Clostridium tyrobutyricum were screened as potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and ferrous sulfate, and these were used in the formulation of a co-culture medium specifically for these two strains. The final medium formula was as follows: peptone 10g / L, beef extract 10g / L, yeast extract 3g / L, glucose 5g / L, sodium chloride 5g / L, soluble starch 1g / L, sodium acetate 3g / L, L-cysteine hydrochloride 0.5g / L, xylan 5g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate heptahydrate 0.6g / L, and ferrous sulfate 0.03g / L.

[0059] Example 3: Co-fermentation characteristics of Lactobacillus plantarum C0502 and Clostridium tyrobutyricum

[0060] After activation, Lactobacillus plantarum and Clostridium tyrobutyricum were inoculated with 1% v / v Lactobacillus plantarum, 1% v / v Clostridium tyrobutyricum or 0.5% v / v Lactobacillus plantarum and 0.5% v / v Clostridium tyrobutyricum respectively into the modified RCM medium, and samples were taken every 4 h or 8 h to detect OD 600 , pH, substrate glucose and fermentation products (lactic acid, acetic acid and butyric acid).

[0061] The experimental results showed that Lactobacillus plantarum C0502 entered the logarithmic growth phase after 4 h of fermentation in the modified RCM ( Figure 4 ), the fermentation substrate glucose begins to be consumed rapidly ( Figure 6 After 16 hours of fermentation, the bacteria entered a stable growth phase, glucose was completely consumed, and the fermentation pH reached its lowest point, stabilizing at around 4.35. At this time, the lactic acid production also basically reached its maximum value (5.60 g / L, Figure 5 and Figure 7 After 16 hours of fermentation, Clostridium tyrobutyricum began to enter the logarithmic growth phase and entered the stable growth phase after 36 hours of fermentation ( Figure 4 Glucose also starts to be consumed rapidly after 16 hours of fermentation and is completely consumed after 40 hours of fermentation ( Figure 6It is worth noting that the pH value of Clostridium tyrobutyricum reached its lowest point after 28h of fermentation in the modified RCM and stabilized at around 5.23, while its butyric acid production reached its maximum value (1.72g / L) at 36h of fermentation ( Figure 5 and Figure 8 ).

[0062] In the co-culture system of Lactobacillus plantarum C0502 and Clostridium tyrobutyricum, the cell concentration (OD 600 ) and glucose consumption in the first 32 h of fermentation were basically consistent with those in the culture of Lactobacillus plantarum C0502 ( Figure 4 Subsequently, the co-culture system entered the second logarithmic growth phase and almost reached its maximum value at 48h of fermentation ( Figure 4 Compared with the single culture of Clostridium tyrobutyricum, the growth of Clostridium tyrobutyricum in the mixed culture system was inhibited to a certain extent, and its time to enter the logarithmic growth phase was significantly delayed by 16h ( Figure 4 During the co-fermentation process, as Clostridium tyrobutyricum entered the logarithmic growth phase at 32 h of fermentation, the pH value of the system increased significantly to 5.29 and remained stable thereafter ( Figure 5 At the same time, the concentrations of lactic acid and acetic acid in the co-fermentation system began to decrease at 32h of fermentation and were close to 0g / L at 60h of fermentation ( Figure 9 In contrast, the butyrate concentration in the co-culture system gradually increased from 32 h of fermentation and reached a maximum of approximately 5.01 g / L at 60 h of fermentation ( Figure 9 ).

[0063] The results showed that Clostridium tyrobutyricum has the ability to convert lactic acid and acetic acid into butyric acid. By comparison, the butyric acid production of Clostridium tyrobutyricum co-cultured with Lactobacillus plantarum C0502 in the modified RCM was 4.58 times and 2.67 times that of Clostridium tyrobutyricum cultured alone in RCM and modified RCM, respectively. Figure 1 , Figure 8 and Figure 9 The results showed that co-cultivation with Lactobacillus plantarum C0502 significantly increased butyrate production.

[0064] The above shows and describes the basic principles, main features and advantages of the present invention, but the invention is not limited to the embodiments. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention, and these changes and improvements fall within the scope of the present invention to be protected.

Claims

1. A method for cultivating butyrate probiotics, characterized in that: The butyric acid probiotic culture medium used includes the following components: 6-12 g / L peptone, 6-12 g / L beef extract, 1-5 g / L yeast extract, 4-8 g / L glucose, 4-8 g / L sodium chloride, 0.5-2 g / L soluble starch, 1-5 g / L sodium acetate, 0.1-1.0 g / L L-cysteine hydrochloride, 4-8 g / L xylan, 1-3 g / L dipotassium hydrogen phosphate, 0.1-1.0 g / L magnesium sulfate heptahydrate, and 0.01-0.05 g / L ferrous sulfate. The culturing method of the butyric acid probiotic comprises the following steps: S1: Lactobacillus plantarum and Clostridium tyrobutyricum bacterial culture solutions were respectively mixed with 40-60% v / v sterile glycerol and frozen to obtain frozen solutions of the two strains. Appropriate amounts of glycerol frozen solutions of Lactobacillus plantarum and Clostridium tyrobutyricum were respectively added to 20-30 volumes of MRS medium and RCM medium, respectively. The cultures were pre-cultured at 35-40°C under anaerobic conditions, wherein Lactobacillus plantarum was cultured for 20-30 hours and Clostridium tyrobutyricum was cultured for 40-55 hours to obtain pre-culture solutions of Lactobacillus plantarum and Clostridium tyrobutyricum; S2: Take appropriate amounts of the pre-cultured liquid of the above-mentioned Lactobacillus plantarum and Clostridium tyrobutyricum, add 8-10 times the volume of MRS medium and RCM medium, respectively, and culture at 35-40°C anaerobically, wherein Lactobacillus plantarum is cultured for 10-15 hours and Clostridium tyrobutyricum is cultured for 30-40 hours to obtain a bacterial suspension at the end of the logarithmic growth phase, wash, and add sterile water to obtain a bacterial suspension with an OD600 of 0.5-0.8; the inoculum size of the Lactobacillus plantarum is 0.5-2% v / v; the inoculum size of the Clostridium tyrobutyricum is 0.5-2% v / v; S3: inoculating the bacterial suspensions of Lactobacillus plantarum and Clostridium tyrobutyricum into any of the butyric acid probiotic culture media and fermenting for 32 to 60 hours; The plant lactobacillus is plant lactobacillus C0502, which is taxonomically named Lactiplantibacillus plantarum, has been deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 31485; The Clostridium tyrobutyricum is Clostridium tyrobutyricum ATCC25755.

2. The butyrate probiotics cultivation method according to claim 1, characterized in that: In S2, the inoculum amount of the Lactobacillus plantarum is 1% v / v.

3. The butyrate probiotics cultivation method according to claim 1, characterized in that: In S2, the inoculation amount of Clostridium tyrobutyricum is 1% v / v.

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