Composite probiotic system for high-yield butyric acid and preparation method thereof

Abstract

The invention provides a butyric acid probiotic culture medium which comprises 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 phosphate, 0.1-1.0 g / L of magnesium sulfate heptahydrate and 0.01-0.05 g / L of ferrous sulfate. The invention specifically provides a method for culturing a lactobacillus plantarum and clostridium tyrobutyricum high-yield butyric acid collaborative system by using the culture medium, and the method not only improves the butyric acid yield, but also reduces the production cost of butyric acid biotransformation, and has remarkable 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 the health of the body, such as providing energy to colon cells, regulating intestinal homeostasis, and regulating intestinal inflammation. A large number of 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 be used as a potential biomarker to assess the health of an organism. Therefore, butyrate is an important physiologically active substance, and it is of great significance to develop a safe, low-cost, and efficient method for producing butyrate.

[0003] At present, butyric acid is mainly derived from chemical synthesis and microbial fermentation, among which most of the commercially available butyric acid is mainly derived from chemical synthesis. Commonly used chemical synthesis methods for producing butyric acid include propylene carbonyl synthesis and n-butyraldehyde oxidation. However, the production of butyric acid by chemical synthesis not only consumes fossil raw materials, but also produces some by-products that are harmful to the environment, such as toxic waste metal catalyst sulfides, cyanides, chlorides, etc.

[0004] Compared with chemically synthesized 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 use 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 some studies have shown that Clostridium tyrobutyricum is a butyric acid-producing bacterium with great development and application value, it is still lacking in research on the efficient utilization of the butyric acid metabolic capacity of Clostridium tyrobutyricum due to its limitations in fermentation substrates (carbon sources) and low butyric acid production. 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 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] Further, 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] The 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 and mixing them with 40-60% v / v sterile glycerol respectively, and freezing and preserving them to obtain the freezing solutions of the two strains; taking appropriate amounts of the glycerol freezing 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 the pre-culture solutions of Lactobacillus plantarum and Clostridium tyrobutyricum;

[0009] 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 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 liquid 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 Lactobacillus plantarum and Clostridium tyrobutyricum into any of the butyric acid probiotic culture media, and fermenting for 4 to 65 hours; further, fermenting for 32 to 60 hours;

[0011] Further, the plant lactobacillus is plant lactobacillus C0502, taxonomically named Lactiplantibacillus plantarum, which has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, the address of which is: No. 3, Yard No. 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] Further, in S2, the inoculation amount of the Lactobacillus plantarum is 0.5-2% v / v; further, the inoculation amount of the Lactobacillus plantarum is 1% v / v;

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

[0015] Figure 1 The effect of different culture methods on butyric acid production.

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

[0017] Figure 3 The pH changes in RCM medium with different culture methods and chemical components are shown;

[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 C. tyrobutyricum in the final medium;

[0023] Fig. 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] The present invention screened out probiotics Lactobacillus plantarum C0502 and Clostridium tyrobutyricum ATCC25755 that can produce high butyric acid through experiments. Clostridium tyrobutyricum ATCC25755 has two main butyric acid metabolism systems: glucose-butyric acid and lactic acid / acetic acid-butyric acid; Lactobacillus plantarum C0502 can be promoted to grow and metabolize lactic acid by cheap biomass raw materials (xylan).

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

[0027] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods, but these embodiments shall not be used to interpret the limitation 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 all fall within the scope defined by the claims of the present invention.

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

[0029] (ii) Culture medium:

[0030] 1. Lactic acid bacteria liquid medium (MRS): 10g / L peptone, 20g / L glucose, 10g / L beef extract, 5g / L yeast powder, 1g / L Tween 80, 2g / L dipotassium hydrogen phosphate, 5g / L sodium acetate, 2g / L sodium citrate, 0.2g / L magnesium sulfate heptahydrate, 0.054g / L manganese sulfate pentahydrate, used for the culture and counting 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, used for the culture and enumeration of Clostridium.

[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] (III) Strains:

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

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

[0036] (IV) Embodiment

[0037] Embodiment 1: Detection method of each index

[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 treatment: Add 0.25 mL of 150 g / L K to 1 mL of sample. 4 Fe(CN) 6 and 0.25mL 300g / L ZnSO 4 After being mixed thoroughly, the mixture was allowed to stand for 30 min. Subsequently, the mixture was centrifuged at 10,000 g for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane.

[0042] 1.2.2 Liquid phase conditions: Instrument: HPLC (Vanquish, Thermo Fisher Scientific, USA); Chromatographic column: C 18 Column (250 mm × 4.6 mm, 5 μm, Thermo Fisher Scientific, USA); Mobile phase: A: chromatographic grade methanol; B: 50 mmol / L K 2 HPO 4(Adjust pH to 2.8 with phosphoric acid); Gradient elution conditions: start with potassium dihydrogen phosphate: methanol = 95:5, perform isocratic elution for 7 minutes, increase methanol concentration linearly from 7 to 16 minutes, reach potassium dihydrogen phosphate: methanol = 60:40 at 16 minutes, and then perform isocratic elution with potassium dihydrogen phosphate: methanol = 60:40, all the above ratios are volume ratios; injection volume: 10 μL; flow rate: 1.0 mL / min; column temperature: 40°C; detector: ultraviolet detector (UV, Thermo Fisher Scientific); detection wavelength: 210 nm.

[0043] 1.3 Detection of glucose by high performance liquid chromatography (HPLC)

[0044] 1.3.1 Sample treatment: Take 1 mL of sample and centrifuge at 10000g for 5 minutes. Filter the supernatant 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 the glycerol frozen stock of Lactobacillus plantarum C0502 and add it to 4.5 mL of MRS medium. Pre-culture it at 37°C under anaerobic conditions for 24 h. Then take 0.5 mL of the pre-cultured bacterial solution and add it to 4.5 mL of MRS medium. Culture it at 37°C under anaerobic conditions for 12 h to obtain the bacterial solution at the end of the logarithmic growth phase. After washing twice, add sterile water to obtain the OD 600 =0.6 bacterial suspension. These bacterial suspensions are used 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. Pre-culture it at 37 °C under anaerobic conditions for 48 h. Then take 0.5 mL of pre-cultured bacterial solution and add it to 4.5 mL RCM medium. Culture it at 37 °C under anaerobic conditions for 36 h to obtain bacterial solution at the end of the logarithmic growth phase. After washing twice, add sterile water and obtain OD 600 =0.6 bacterial suspension. These bacterial suspensions are used as seed liquid for subsequent fermentation.

[0054] 2.3 Optimization of co-fermentation medium

[0055] The culture medium includes (a) RCM; (b) RCM + 5g / 100mL xylan; (c) RCM + 5g / 100mL xylan + 2g / L potassium dihydrogen phosphate; (d) RCM + 5g / 100mL xylan + 2g / L sodium citrate; (e) RCM + 5g / 100mL xylan + 0.6g / L magnesium sulfate heptahydrate; (f) RCM + 5g / 100mL xylan + 0.054g / L manganese sulfate pentahydrate; (g) RCM + 5g / 100mL xylan + 3g / L ammonium sulfate; (h) RCM + 5g / 100mL xylan + 0.03g / L ferrous sulfate; (i) RCM + 5g / 100mL xylan + 40g / 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 3 As 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 mono-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 can significantly increase 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 butyric acid production in the co-culture system of Lactobacillus plantarum C0502 and Clostridium tyrobutyricum compared with the culture in the RCM medium containing xylan ( Figure 2 ).

[0058] Therefore, the components that effectively stimulate butyric acid production in the co-culture system of Lactobacillus plantarum C0502 and Clostridium tyrobutyricum were screened out as potassium dihydrogen phosphate, magnesium sulfate heptahydrate and ferrous sulfate, and they were used in the formula of the special co-culture medium for these two strains. The final culture 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, 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 in modified RCM medium, and samples were taken every 4h or 8h 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 the lowest level, stabilizing at around 4.35. At this time, the lactic acid production also basically reached the maximum value (5.60 g / L, Figure 5 and Figure 7 ). After 16 hours of fermentation with modified RCM, 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 6 ). It is worth noting that the pH value of Clostridium tyrobutyricum reached the lowest point after 28h of fermentation in the modified RCM, stabilizing at around 5.23, while its butyric acid production reached the 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 the 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 the time for it 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 ( Fig. 9 ). In contrast, the butyrate concentration in the co-culture system gradually increased from 32h of fermentation and reached a maximum value of about 5.01g / L at 60h of fermentation ( Fig. 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 Fig. 9 ). This result indicates that co-cultivation with Lactobacillus plantarum C0502 significantly increased butyrate production.

[0064] The basic principles, main features and advantages of the present invention are shown and described above, but the present 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 all fall within the scope of the present invention to be protected.

Claims

1. A butyrate probiotic culture medium, It is characterized in that The invention comprises 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.

2. The butyrate probiotic culture medium according to claim 1, It is characterized in that The butyrate probiotic culture medium comprises 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.

3. A method for culturing butyrate probiotics, It is characterized in that The following steps are involved: S1: taking Lactobacillus plantarum and Clostridium tyrobutyricum bacterial culture solutions and mixing them with 40-60% v / v sterile glycerol respectively, and freezing and preserving them to obtain the freezing solutions of the two strains; taking appropriate amounts of the glycerol freezing 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 the 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 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 liquid 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; S3: inoculating the bacterial suspension Lactobacillus plantarum and Clostridium tyrobutyricum into any of the butyric acid probiotic culture media, and fermenting for 4 to 65 hours; further, fermenting for 32 to 60 hours.

4. The butyrate probiotics culturing method according to claim 3, It is characterized in that The plant lactobacillus is plant lactobacillus C0502, which is taxonomically named Lactiplantibacillus plantarum and has been deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No.31485.

5. The butyrate probiotics culturing method according to claim 3, It is characterized in that The Clostridium tyrobutyricum is Clostridium tyrobutyricum ATCC25755.

6. The butyrate probiotics culturing method according to claim 3, It is characterized in that The inoculation amount of the Lactobacillus plantarum is 0.5-2% v / v; further, the inoculation amount of the Lactobacillus plantarum is 1% v / v.

7. The butyrate probiotics culturing method according to claim 3, It is characterized in that The inoculation amount of Clostridium tyrobutyricum is 0.5-2% v / v; further, the inoculation amount of Lactobacillus plantarum is 1% v / v).

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