A method for co-fermentation of Clostridium butyricum and Lactobacillus amylophilus

Abstract

The present invention relates to the field of biotechnology. Specifically, it relates to a co-fermentation method of Clostridium butyricum and Lactobacillus amylovorans. The co-fermentation method uses a compound preparation, and the compound preparation contains a Lactobacillus amylovorans and Clostridium butyricum. The Lactobacillus amylovorans is Lactobacillus amylovorus, and its strain number is RZ-D4L1, which is preserved in the China Center for Type Culture Collection, and the preservation number is: CCTCC NO: M 2024761; the Clostridium butyricum HZNDC-1 is isolated from the large intestine of cattle, and is preserved in the China Center for Type Culture Collection, and the preservation number is: CCTCC NO: M 2021874. The co-fermentation of the present invention can improve the fermentation stability of Clostridium butyricum, and at the same time, there is also a significant increase in the viable cell count and sporulation rate of Clostridium butyricum, providing a new fermentation method for the high-density fermentation of Clostridium butyricum, which can increase the probiotic performance of the fermentation supernatant, improve the utilization value of the waste liquid of Clostridium butyricum, and has great popularization and application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a method for co-fermentation of Clostridium butyricum and Lactobacillus amylovorus. Background Art

[0002] Clostridium butyricum, also known as butyric acid bacteria, Clostridium butyricum bacillus, and butyric acid bacteria, is a strictly anaerobic Gram-positive spore-forming bacillus. Due to its ability to form spores, Clostridium butyricum has characteristics such as high temperature resistance, gastric acid resistance, bile salt resistance, and resistance to some antibiotics, making it an excellent strain of new feed microorganisms after Bacillus, yeast, and lactic acid bacteria.

[0003] Clostridium butyricum is usually used as a feed additive in the aquaculture industry and can exhibit good biological properties in the animal intestine: producing acidic substances such as short-chain fatty acids, repairing the intestinal mucosa, and reducing the intestinal pH; inhibiting the reproduction of harmful bacteria, promoting the proliferation and development of beneficial intestinal flora in animals, and regulating the balance of intestinal flora; synthesizing various digestive enzymes and nutrients to provide nutrients for the body. Clostridium butyricum has a wide range of biological activities and is widely used as an antibiotic substitute in animal production, aquaculture and other fields as a feed additive.

[0004] At present, Clostridium butyricum has a wide market application, but due to problems such as low fermentation cell count and unstable fermentation culture, the production cost remains high, thus limiting the application of Clostridium butyricum in the livestock industry. Existing technologies include batch continuous fermentation of Clostridium butyricum, which increases the process flow, has a long fermentation cycle, and the fermentation density does not increase significantly; there are also optimizations of Clostridium butyricum fermentation parameters such as stirring speed and sugar concentration regulation, which have no adaptability for different Clostridium butyricum fermentation parameters and cannot completely solve the problem of unstable fermentation.

[0005] Therefore, providing a solution that can achieve stable fermentation of Clostridium butyricum is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] The present invention first provides a Lactobacillus amylovorus, which is Lactobacillus amylovorus, with the strain number RZ-D4L1, and it is preserved in the China Center for Type Culture Collection, with the preservation number: CCTCC NO: M 2024761.

[0007] The present invention also provides a compound preparation, which contains the above-mentioned Lactobacillus amylovorus and Clostridium butyricum. The Clostridium butyricum HZNDC-1 is isolated from the large intestine of cattle and is preserved in the China Center for Type Culture Collection, with the preservation number: CCTCC NO: M 2021874.

[0008] In some embodiments, the viable count of Clostridium butyricum is 1.0×10 6 cfu / mL; the viable count of Lactobacillus amylovorus is 1.0×10 3 cfu / mL to 1.0×10 6 cfu / mL.

[0009] In some embodiments, the viable count of Lactobacillus amylovorus is 1.0×10 4 cfu / mL.

[0010] The present invention also provides the application of the above compound preparation in fermenting butyric acid and lactic acid.

[0011] The present invention also provides the application of the above compound preparation in increasing the viable count and sporulation rate of Clostridium butyricum.

[0012] The present invention also provides a co-fermentation method of Clostridium butyricum and Lactobacillus amylovorus, and the fermentation method includes the step of inoculating the above compound preparation.

[0013] In some embodiments, the formula for the co-fermentation is: beef extract powder 20 g / L, peptone 20 g / L, yeast extract powder 15 g / L, glucose 10 g / L, soluble starch 8 g / L, potassium dihydrogen phosphate 1.3 g / L, magnesium sulfate heptahydrate 1.2 g / L, sodium chloride 1 g / L, L-cysteine hydrochloride 0.5 g / L, pH 6.8±0.1.

[0014] In some embodiments, the soybean meal fermentation adopts a solid-state fermentation method. After inoculating the above compound preparation, nitrogen is introduced to replace the air in the fermentation tank, and it is sealed. The stirring speed is controlled at 100 rpm, and the temperature is 35°C to 37°C. After 4 h of fermentation culture, at this time, Lactobacillus amylovorus RZ-D4L1 can enter the fermentation tank of Clostridium butyricum HZNDC-1 at a flow rate of 400 ml / h for continuous fermentation in the same fermentation tank. Nitrogen is introduced again to replace the air in the fermentation tank, and it is sealed. It is cultured at 37°C and a stirring speed of 150 rpm for 48 h.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects:

[0016] 1. The co-fermentation of Clostridium butyricum and Lactobacillus amylovorus can improve the fermentation stability of Clostridium butyricum, and at the same time, there is also an obvious increase in the viable count and sporulation rate of Clostridium butyricum, providing a new fermentation method for the high-density fermentation of Clostridium butyricum.

[0017] 2. Clostridium butyricum can metabolize lactic acid during the fermentation process. As an intermediate metabolite of Clostridium butyricum, lactic acid will be decomposed and utilized as an energy source along with the subsequent growth of Clostridium butyricum. Therefore, the co-fermentation of Clostridium butyricum and Lactobacillus amylovorans will increase the lactic acid content during the early growth of Clostridium butyricum, stimulate the growth of Clostridium butyricum to achieve the purpose of high-density fermentation, and increase the viable count of Clostridium butyricum.

[0018] 3. The fermentation supernatant produced by the co-fermentation of Clostridium butyricum and Lactobacillus amylovorans is not only rich in butyric acid and other metabolites of Clostridium butyricum, but also increases the content of lactic acid and other metabolites of Lactobacillus amylovorans, which can increase the probiotic performance of the fermentation supernatant, improve the utilization value of the waste liquid of Clostridium butyricum, and has great popularization and application value.

[0019] Depositing Instructions

[0020] The Clostridium butyricum HZNDC-1 was isolated from bovine intestine and sent to the China Center for Type Culture Collection for deposit on July 13, 2021. It was taxonomically named Clostridium butyricum HZNDC-1, and the deposit number was CCTCC: NO: M 2021874, with the address: Wuhan University, Wuhan, Hubei, China. See CN115094010 B.

[0021] The Lactobacillus amylovorus RZ-D4L1 was isolated from porcine intestinal mucus and sent to the China Center for Type Culture Collection for deposit on April 24, 2024. It was taxonomically named Lactobacillus amylovorus RZ-D4L1 and deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2024761, and the address: Wuhan University, Wuhan, Hubei, China. Detailed Embodiments

[0022] To make the technical problems, technical solutions, and advantages to be solved by the present invention clearer, the following will be described in detail with specific embodiments.

[0023] Example 1: Comparison of Co-Cultivation of Clostridium butyricum HZNDC-1 with Different Lactic Acid Bacteria

[0024] Preparation of Clostridium butyricum Seed Solution:

[0025] Clostridium butyricum seed liquid medium (RCM): Beef extract powder 10 g / L, peptone 10 g / L, yeast extract powder 3 g / L, glucose 5 g / L, soluble starch 1 g / L, sodium chloride 5 g / L, sodium acetate 5 g / L, L-cysteine hydrochloride 0.5 g / L, pH 6.8 ± 0.1, sterilized at 115 °C for 30 min for standby use.

[0026] Pick a loop of purified single colony of Clostridium butyricum HZNDC-1 and inoculate it into the RCM liquid medium, and culture it in an anaerobic workstation at 37 °C for 24 h to prepare a seed liquid.

[0027] Preparation of lactic acid bacteria seed liquid:

[0028] Lactic acid bacteria seed liquid medium (MRS): Beef extract powder 10 g / L, peptone 10 g / L, yeast extract powder 5 g / L, glucose 20 g / L, sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, Tween-80 1 mL / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate heptahydrate 0.05 g / L, pH 6.3 ± 0.1, sterilized at 121 °C for 15 min for standby use.

[0029] Pick a loop of purified single colony of lactic acid bacteria and inoculate it into the RCM liquid medium, and culture it in a constant temperature incubator at 37 °C for 24 h to prepare the corresponding seed liquid.

[0030] Co-culture of Clostridium butyricum HZNDC-1 and different lactic acid bacteria:

[0031] Co-culture evaluation medium: Beef extract powder 10 g / L, peptone 10 g / L, yeast extract powder 3 g / L, glucose 5 g / L, soluble starch 1 g / L, sodium chloride 5 g / L, L-cysteine hydrochloride 0.5 g / L, pH 6.8 ± 0.1, sterilized at 115 °C for 30 min for standby use.

[0032] Inoculate the activated Clostridium butyricum HZNDC-1 seed liquid and 12 kinds of activated lactic acid bacteria seed liquids into the co-culture evaluation medium at an inoculation amount of 1% respectively, and culture them in an anaerobic workstation at 37 °C for 24 h; at the same time, set a blank control group inoculated with Clostridium butyricum alone, and set 3 replicates for all groups. Measure data such as lactic acid, viable count of Clostridium butyricum, and sporulation rate of Clostridium butyricum. The results are shown in Table 1 below:

[0033] The measurement indexes of the co-culture of Clostridium butyricum HZNDC-1 and different lactic acid bacteria are shown in Table 1 below:

[0034] Table 1 Comparison of co-culture indexes of Clostridium butyricum HZNDC-1 and different lactic acid bacteria

[0035]

[0036]

[0037] Note: The data in the table are average values.

[0038] As can be seen from Table 1, there are significant differences in the effects of co-culturing different lactic acid bacteria with Clostridium butyricum on the performance of Clostridium butyricum HZNDC-1. Among them, the strain numbered 10 showed the best performance among the 12 lactic acid bacteria evaluated. The strain No. 10 is Lactobacillus amylovorus RZ-D4L1 isolated from porcine intestinal mucus and is deposited in the China Center for Type Culture Collection with the deposit number: CCTCC NO: M2024761. This indicates that Lactobacillus amylovorus RZ-D4L1 grows synergistically with Clostridium butyricum HZNDC-1, thus significantly increasing the viable cell count and sporulation rate of Clostridium butyricum HZNDC-1.

[0039] Example 2: Co-fermentation production method of Clostridium butyricum HZNDC-1 and Lactobacillus amylovorus RZ-D4L1

[0040] In order to explore the effects of different inoculation amounts of Clostridium butyricum HZNDC-1 and Lactobacillus amylovorus RZ-D4L1 on the fermentation effect of Clostridium butyricum, the following comparative experiments were designed in the table:

[0041] Table 2 Design of different inoculation amounts of Clostridium butyricum HZNDC-1 and Lactobacillus amylovorus

[0042] Clostridium butyricum HZNDC-1 (cfu / mL) Lactobacillus amylovorus RZ-D4L1 (cfu / mL) Inoculum amount 1 <![CDATA[1.0×10 6 > <![CDATA[1.0×10 5 > Inoculum amount 2 <![CDATA[1.0×10 6 > <![CDATA[1.0×10 4 > Inoculum amount 3 <![CDATA[1.0×10 6 > <![CDATA[1.0×10 3 > Control group <![CDATA[1.0×10 6 > /

[0043] The specific operation steps are as follows:

[0044] Preparation of Clostridium butyricum HZNDC-1 seed liquid:

[0045] Clostridium butyricum seed liquid medium (RCM): Beef extract powder 10 g / L, peptone 10 g / L, yeast extract powder 3 g / L, glucose 5 g / L, soluble starch 1 g / L, sodium chloride 5 g / L, sodium acetate 5 g / L, L-cysteine hydrochloride 0.5 g / L, pH 6.8 ± 0.1, sterilized at 115 °C for 30 min for standby.

[0046] Pick a loop of purified single colonies of Clostridium butyricum HZNDC-1 and inoculate them into the RCM liquid medium, and culture them in an anaerobic workstation at 37 °C for 24 h to prepare the seed liquid.

[0047] Preparation of Lactobacillus amylovorus RZ-D4L1 seed liquid:

[0048] Lactic acid bacteria seed liquid medium (MRS): Beef extract powder 10 g / L, peptone 10 g / L, yeast extract powder 5 g / L, glucose 20 g / L, sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, Tween-80 1 mL / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate heptahydrate 0.05 g / L, pH 6.3 ± 0.1, sterilized at 121 °C for 15 min for standby.

[0049] Pick a loop of purified single colony of Lactobacillus amylophilus RZ-D4L1 and inoculate it into RCM liquid medium, place it in a constant temperature incubator at 37 °C for 24 h to prepare the corresponding seed liquid.

[0050] Synergistic fermentation of Clostridium butyricum HZNDC-1 and Lactobacillus amylophilus RZ-D4L1:

[0051] Fermentation medium preparation

[0052] Fermentation medium: Beef extract powder 20 g / L, peptone 20 g / L, yeast extract powder 15 g / L, glucose 10 g / L, soluble starch 8 g / L, potassium dihydrogen phosphate 1.3 g / L, magnesium sulfate heptahydrate 1.2 g / L, sodium chloride 1 g / L, L-cysteine hydrochloride 0.5 g / L, pH 6.8 ± 0.1.

[0053] Fermentation tank A (5 L): 1.5 L of fermentation medium, sterilized at 121 °C for 30 min for standby.

[0054] Fermentation tank B (5 L): 1.5 L of fermentation medium, sterilized at 121 °C for 30 min for standby.

[0055] Fermentation culture

[0056] Inoculate the seed liquid of Clostridium butyricum HZNDC-1 into fermentation tank A at the corresponding inoculation amount, introduce nitrogen to displace the air in the fermentation tank, seal it, and culture it at 37 °C and a stirring speed of 100 rpm; at the same time, inoculate the seed liquid of Lactobacillus amylophilus RZ-D4L1 into fermentation tank B at the corresponding inoculation amount, introduce nitrogen to displace the air in the fermentation tank, seal it, and culture it at 37 °C and a stirring speed of 100 rpm.

[0057] After 2 h of fermentation culture, introduce the fermentation broth in fermentation tank B into fermentation tank A. At this time, Clostridium butyricum HZNDC-1 and Lactobacillus amylophilus RZ-D4L1 can be mixed and continuously fermented in fermentation tank A. Introduce nitrogen again to displace the air in the fermentation tank, seal it, and culture it at 37 °C and a stirring speed of 100 rpm for 48 h, and then measure data such as lactic acid, butyric acid, viable count of Clostridium butyricum, and sporulation rate of Clostridium butyricum.

[0058] The results are shown in Table 3 below:

[0059] Table 3 Comparison of fermentation indexes of different inoculation amounts between Clostridium butyricum HZNDC-1 and Lactobacillus amylophilus RZ-D4L1

[0060]

[0061] Note: The data in the table are the averages after drying to constant weight.

[0062] As can be seen from Table 3, the combination of Clostridium butyricum HZNDC-1 and Lactobacillus amylophilus RZ-D4L1 can promote the high-density fermentation of Clostridium butyricum, and the effect is better than using any one of them alone. And within the tested range, the indexes of inoculation amount 2 are the best.

[0063] In addition, the fermentation broth produced by the co-fermentation of Clostridium butyricum HZNDC-1 and Lactobacillus amylophilus RZ-D4L1 significantly increased the contents of butyric acid and lactic acid, and increased the activity of the fermentation broth.

[0064] Example 3: Co-fermentation production method of Clostridium butyricum HZNDC-1 and Lactobacillus amylophilus RZ-D4L1

[0065] In order to explore the effect of inoculating Lactobacillus amylophilus RZ-D4L1 at different fermentation times of Clostridium butyricum HZNDC-1 on the fermentation effect of Clostridium butyricum, the following comparative experiments were designed:

[0066] Table 4 Time design of co-fermentation by inoculating Lactobacillus amylophilus RZ-D4L1 in the fermentation tank of Clostridium butyricum HZNDC-1

[0067]

[0068]

[0069] The specific operation steps are as follows:

[0070] Preparation of Clostridium butyricum HZNDC-1 seed liquid:

[0071] Clostridium butyricum seed liquid medium (RCM): Beef extract powder 10 g / L, peptone 10 g / L, yeast extract powder 3 g / L, glucose 5 g / L, soluble starch 1 g / L, sodium chloride 5 g / L, sodium acetate 5 g / L, L-cysteine hydrochloride 0.5 g / L, pH 6.8 ± 0.1, sterilized at 115 °C for 30 min for standby.

[0072] Pick a loop of purified single colony of Clostridium butyricum HZNDC-1 and inoculate it into the RCM liquid medium, and culture it in an anaerobic workstation at 37 °C for 24 h to make a seed liquid.

[0073] Preparation of Lactobacillus amylophilus RZ-D4L1 seed liquid:

[0074] Lactic acid bacteria seed liquid medium (MRS): Beef extract powder 10 g / L, peptone 10 g / L, yeast extract powder 5 g / L, glucose 20 g / L, sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, Tween-80 1 mL / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate heptahydrate 0.05 g / L, pH 6.3 ± 0.1, sterilized at 121 °C for 15 min and reserved for use.

[0075] Pick a purified single colony of Lactobacillus amylophilus RZ-D4L1 and inoculate it into RCM liquid medium, and culture it in a constant temperature incubator at 37 °C for 24 h to prepare the corresponding seed liquid.

[0076] Synergistic fermentation of Clostridium butyricum HZNDC-1 and Lactobacillus amylophilus RZ-D4L1:

[0077] Preparation of fermentation medium

[0078] Fermentation medium: Beef extract powder 20 g / L, peptone 20 g / L, yeast extract powder 15 g / L, glucose 10 g / L, soluble starch 8 g / L, potassium dihydrogen phosphate 1.3 g / L, magnesium sulfate heptahydrate 1.2 g / L, sodium chloride 1 g / L, L-cysteine hydrochloride 0.5 g / L, pH 6.8 ± 0.1.

[0079] Fermentation tank A (5 L): 1.5 L of fermentation medium, sterilized at 121 °C for 30 min and reserved for use.

[0080] Fermentation tank B (5 L): 1.5 L of fermentation medium, sterilized at 121 °C for 30 min and reserved for use.

[0081] Fermentation culture

[0082] Inoculate the seed liquid of Clostridium butyricum HZNDC-1 into fermentation tank A at 1.0×10 6 cfu / mL, introduce nitrogen to displace the air in the fermentation tank, seal it, and culture it at 37 °C and a stirring speed of 100 rpm; at the same time, inoculate the seed liquid of Lactobacillus amylophilus RZ-D4L1 into fermentation tank B at 1.0×10 4 cfu / mL, introduce nitrogen to displace the air in the fermentation tank, seal it, and culture it at 37 °C and a stirring speed of 100 rpm.

[0083] After fermenting and culturing for the corresponding time, introduce the fermentation broth in fermentation tank B into fermentation tank A. At this time, Clostridium butyricum HZNDC-1 and Lactobacillus amylophilus RZ-D4L1 can be mixed and continuously fermented in fermentation tank A. Then introduce nitrogen again to displace the air in the fermentation tank, seal it, and culture it at 37 °C and a stirring speed of 100 rpm for 48 h, and then measure data such as the viable count of Clostridium butyricum and the sporulation rate of Clostridium butyricum.

[0084] The results are shown in Table 5 below:

[0085] Table 5 Data comparison of co-fermentation by inoculating Lactobacillus amylovorus RZ-D4L1 into the Clostridium butyricum HZNDC-1 fermenter at different fermentation times

[0086]

[0087] Note: The data in the table are the averages after drying to a constant weight.

[0088] As can be seen from Table 4, when Lactobacillus amylovorus RZ-D4L1 is inoculated too quickly into the Clostridium butyricum fermenter for co-fermentation, the growth rate of Lactobacillus amylovorus RZ-D4L1 may be too fast, resulting in excessive lactic acid accumulation, and the normal growth of Clostridium butyricum may be affected. When Lactobacillus amylovorus RZ-D4L1 is inoculated too slowly into the Clostridium butyricum fermenter for co-fermentation, nutritional competition may occur, and the growth of Clostridium butyricum is restricted, and the final cell count cannot be significantly increased.

[0089] Within the tested range, it shows that after inoculating Lactobacillus amylovorus RZ-D4L1 into the Clostridium butyricum HZNDC-1 fermenter for co-fermentation 4 h after inoculation, the fermentation cell count of Clostridium butyricum is significantly increased and the sporulation rate is also improved.

[0090] Example 4: Co-fermentation production method of Clostridium butyricum HZNDC-1 and Lactobacillus amylovorus RZ-D4L1

[0091] In order to explore the influence of the flow rate of the fermentation broth of Lactobacillus amylovorus RZ-D4L1 entering the Clostridium butyricum HZNDC-1 fermenter on the fermentation effect of Clostridium butyricum HZNDC-1 during the co-fermentation of Clostridium butyricum HZNDC-1 and Lactobacillus amylovorus RZ-D4L1, the following comparative experiment was designed:

[0092] Table 6 Design of the flow rate of the fermentation broth of Lactobacillus amylovorus RZ-D4L1 added to the Clostridium butyricum HZNDC-1 fermenter

[0093] Flow rate Experimental group 1 800 ml / h Experimental group 2 600 ml / h Experimental group 3 400 ml / h Control group 1000 ml / h

[0094] The specific operation steps are as follows:

[0095] (1) Preparation of Clostridium butyricum HZNDC-1 seed liquid:

[0096] Clostridium butyricum seed liquid medium (RCM): Beef extract powder 10 g / L, peptone 10 g / L, yeast extract powder 3 g / L, glucose 5 g / L, soluble starch 1 g / L, sodium chloride 5 g / L, sodium acetate 5 g / L, L-cysteine hydrochloride 0.5 g / L, pH 6.8 ± 0.1, sterilized at 115 °C for 30 min and reserved for use.

[0097] Pick a purified single colony of Clostridium butyricum HZNDC-1 and inoculate it into RCM liquid medium. Incubate it in an anaerobic workstation at 37°C for 24 h to prepare a seed solution.

[0098] (2) Preparation of Lactobacillus amylophilus RZ-D4L1 seed solution:

[0099] Lactic acid bacteria seed solution medium (MRS): Beef extract powder 10 g / L, peptone 10 g / L, yeast extract powder 5 g / L, glucose 20 g / L, sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, Tween-80 1 mL / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate heptahydrate 0.05 g / L, pH 6.3 ± 0.1, sterilize at 121°C for 15 min and keep for later use.

[0100] Pick a purified single colony of Lactobacillus amylophilus RZ-D4L1 and inoculate it into RCM liquid medium. Incubate it in a constant temperature incubator at 37°C for 24 h to prepare the corresponding seed solution.

[0101] (3) Co-fermentation of Clostridium butyricum HZNDC-1 and Lactobacillus amylophilus RZ-D4L1:

[0102] (3.1) Preparation of fermentation medium

[0103] Fermentation medium: Beef extract powder 20 g / L, peptone 20 g / L, yeast extract powder 15 g / L, glucose 10 g / L, soluble starch 8 g / L, potassium dihydrogen phosphate 1.3 g / L, magnesium sulfate heptahydrate 1.2 g / L, sodium chloride 1 g / L, L-cysteine hydrochloride 0.5 g / L, pH 6.8 ± 0.1.

[0104] Fermentation tank A (5 L): 1.5 L of fermentation medium, sterilize at 121°C for 30 min and keep for later use.

[0105] Fermentation tank B (5 L): 1.5 L of fermentation medium, sterilize at 121°C for 30 min and keep for later use.

[0106] (3.2) Fermentation culture

[0107] Inoculate the seed solution of Clostridium butyricum HZNDC-1 into fermentation tank A at 1.0×10 6 cfu / mL, introduce nitrogen to displace the air in the fermentation tank, seal it, and culture it at 37°C and a stirring speed of 100 rpm; at the same time, inoculate the seed solution of Lactobacillus amylophilus RZ-D4L1 into fermentation tank B at 1.0×10 4 cfu / mL, introduce nitrogen to displace the air in the fermentation tank, seal it, and culture it at 37°C and a stirring speed of 100 rpm.

[0108] After 4 h of fermentation culture, all the fermentation broth in the B tank of the fermenter was fed into the A tank of the fermenter at a corresponding rate. At this time, Clostridium butyricum HZNDC-1 and Lactobacillus amylophilus RZ-D4L1 could be mixed and continuously fermented in the A tank of the fermenter. Nitrogen was introduced again to displace the air in the fermenter, and then it was sealed. After culturing for 48 h at 37 °C and a stirring speed of 100 rpm, data such as the viable count of Clostridium butyricum and the sporulation rate of Clostridium butyricum were measured.

[0109] Table 7 Data comparison of adding Lactobacillus amylophilus RZ-D4L1 to the fermenter of Clostridium butyricum HZNDC-1 at different feeding rates

[0110]

[0111] As can be seen from the data in Table 6, when Clostridium butyricum HZNDC-1 and Lactobacillus amylophilus RZ-D4L1 were co-fermented, adding Clostridium butyricum to the fermenter at a low flow rate could promote the high-density fermentation of Clostridium butyricum. And within the tested range, the indicators of experimental group 3 were the best, with the viable count as high as 5.34×10 10 cfu / mL and the spore conversion rate of 96.24%.

[0112] The above is the preferred implementation mode of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Lactobacillus amylovora, characterized in that The amylovora lactobacillus is amylovora lactobacillus ( Lactobacillus amylovorus ), the strain number is RZ-D4L1, which is deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M 2024761.

2. A compound preparation, characterized in that: The compound preparation contains the amylovorax lactobacillus and the clostridium butyricum according to claim 1. Clostridium butyricum )HZNDC-1, the Clostridium butyricum ( Clostridium butyricum )HZNDC-1 was isolated from bovine large intestine and deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M2021874.

3. The compound preparation according to claim 2, characterized in that: The viable count of Clostridium butyricum is 1.0×10 6 cfu / mL; the viable count of Lactobacillus amylovora was 1.0×10 3 cfu / mL to 1.0×10 6 cfu / mL.

4. The compound preparation according to claim 2, characterized in that: The viable count of Lactobacillus amylovora is 1.0×10 4 cfu / mL.

5. Use of the compound preparation according to any one of claims 2 to 4 in fermenting butyric acid and lactic acid.

6. Use of the compound preparation according to any one of claims 2 to 4 in increasing the viable count and sporulation rate of Clostridium butyricum.

7. A method for the joint fermentation of Clostridium butyricum and Lactobacillus amylovora, characterized in that: The fermentation method comprises the step of inoculating the compound preparation according to any one of claims 2-4.

8. The method according to claim 7, characterized in that The culture medium formula of the linked fermentation is: 20 g / L beef extract powder, 20 g / L peptone, 15 g / L yeast extract powder, 10 g / L glucose, 8 g / L soluble starch, 1.3 g / L potassium dihydrogen phosphate, 1.2 g / L magnesium sulfate heptahydrate, 1 g / L sodium chloride, 0.5 g / L L-cysteine ​​hydrochloride, pH 6.8±0.1.

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