CN116396874A - Combined fermentation method of clostridium butyricum - Google Patents

Abstract

The invention relates to a combined fermentation method of clostridium butyricum, which comprises the following steps: sequentially and timely inoculating Saccharomyces cerevisiae, lactobacillus acidophilus and Clostridium butyricum into a liquid culture medium, and performing joint fermentation of Clostridium butyricum without additionally providing an anaerobic environment. The combined fermentation method provides a new green energy-saving culture mode, meets the growth requirements of clostridium butyricum, lactobacillus acidophilus and saccharomyces cerevisiae by a time-staggered inoculation method in the combined liquid fermentation process, and combines fermentation productsThe viable count and the spore count of the clostridium butyricum can reach 2.73X10 ⁹ cfu/mL and 2.65X10 ⁹ cfu/mL, effectively solves the problem of lower clostridium butyricum concentration in clostridium butyricum mixed fermentation in the prior art. In addition, the recycling of the combined fermentation centrifugal waste liquid reduces the resource waste caused by wastewater treatment and reduces the cost of related enterprises for fermentation wastewater treatment.

Description

A combined fermentation method of Clostridium butyricum

Technical Field

The invention relates to a combined fermentation method of Clostridium butyricum, and belongs to the technical field of microecological preparation production methods.

Background Art

In recent years, microecological preparations have played an important role in the fields of medicine and health care, food, feed, livestock and poultry breeding, etc. With the gradual implementation of the "antibiotic restriction order", microecological preparations are increasingly used in the field of livestock and poultry breeding. Studies have shown that microecological preparations can maintain the balance of flora in the intestines of animals, prevent intestinal infections, and improve the immunity of animals. In addition, since probiotics can secrete a variety of digestive enzymes, microecological preparations play a icing on the cake role in improving feed utilization, improving animal production performance, and improving the quality of livestock and poultry products.

Clostridium butyricum has a strong intestinal regulating effect. Due to its great advantages in regulating animal intestinal function, improving animal growth performance and enhancing immunity, it is increasingly used in feed additives. Lactobacillus acidophilus belongs to the genus Lactobacillus and is clinically used to regulate the balance of intestinal flora and inhibit the proliferation of unhealthy intestinal microorganisms. Brewer's yeast can promote the absorption of nutrients in feed and improve animal immunity. It is also a nutritious single-cell protein that can shorten the feeding period, improve meat quality and increase lean meat rate.

Clostridium butyricum is a strict anaerobic bacterium. During its fermentation process, it needs to be carried out in an anaerobic environment maintained by paraffin oil or inert gases such as nitrogen and carbon dioxide. This increases the complexity of the equipment and the difficulty of the method during the fermentation process of Clostridium butyricum, and increases the production cost.

To solve the above problems, the Chinese invention patent application with publication number CN111500508A discloses a liquid mixed fermentation method of Clostridium butyricum and Bacillus coagulans, wherein the seed liquid of Bacillus coagulans and Clostridium butyricum is prepared and then inoculated into a liquid culture medium for fermentation. In this mixed fermentation method, the live bacteria count and spore count of Clostridium butyricum can reach 5.8×10 ⁸ cfu/mL and 5.3×10 ⁸ cfu/mL.

The Chinese invention patent application with publication number CN110241053A discloses a method for culturing Clostridium butyricum by mixed fermentation, wherein Clostridium butyricum, Bacillus subtilis, yeast and lactic acid bacteria are first cultured separately to obtain single bacterial culture fluids, and then mixed in a certain proportion for mixed culture. With this mixed fermentation culture method, the concentration of Clostridium butyricum can reach 1×10 ⁹ cfu/mL.

However, the above mixed fermentation methods are all inoculated at the same time, in which Bacillus coagulans and Bacillus subtilis grow fast and consume too much nutrients, which to a certain extent affects the concentration of Clostridium butyricum in the final fermentation product. Therefore, it is an urgent problem to develop a fermentation method that is simple, low-cost, and has a higher concentration of Clostridium butyricum.

Summary of the invention

In order to solve the above problems, the purpose of the present invention is to provide a combined fermentation method of Clostridium butyricum, which avoids the need for nitrogen or anaerobic agents to provide anaerobic conditions during the anaerobic fermentation of Clostridium butyricum, thereby increasing the fermentation cost and equipment investment, and solves the problem of low Clostridium butyricum concentration in the mixed fermentation of Clostridium butyricum in the prior art.

In order to achieve the above object, the technical scheme of the combined fermentation method of Clostridium butyricum in the present invention is:

A Clostridium butyricum co-fermentation method comprises the following steps: inoculating saccharomyces cerevisiae, lactobacillus acidophilus and Clostridium butyricum into a liquid culture medium in sequence at different times, and performing co-fermentation of Clostridium butyricum without providing an additional anaerobic environment.

The beneficial effect of the above technical solution is that: the present invention inoculates saccharomyces cerevisiae, lactobacillus acidophilus and clostridium butyricum into the liquid culture medium at different times for joint fermentation and culture. In the joint fermentation, saccharomyces cerevisiae and lactobacillus acidophilus are sufficient to consume oxygen in the culture medium, provide an anaerobic environment for the culture of clostridium butyricum, and consume less nutrients while completing the proliferation process, so that more nutrients are allocated to clostridium butyricum. In the mixed fermentation liquid obtained after the fermentation is completed, the number of live bacteria and the number of spores of clostridium butyricum can reach 2.73×10 ⁹ cfu/mL and 2.65×10 ⁹ cfu/mL.

As a further improvement, the staggered inoculation is to first inoculate the brewer's yeast into the liquid culture medium, then inoculate the lactobacillus acidophilus 5 to 8 hours later, and then inoculate the clostridium butyricum 12 to 16 hours later. Preferably, the initial pH of the liquid culture medium is 6.8 to 7, the filling coefficient is 40 to 50%, the brewer's yeast is inoculated into the liquid culture medium at an inoculation amount of 4 to 5%, and after culturing at 35 to 37° C. for 5 to 8 hours, the lactobacillus acidophilus is inoculated at an inoculation amount of 2 to 3% to continue culturing, and after 12 to 16 hours, the clostridium butyricum is finally inoculated at an inoculation amount of 4 to 5%.

The beneficial effect of the above technical solution is that Lactobacillus acidophilus is a facultative anaerobic bacterium, which can further consume the oxygen in the system on the basis of yeast, thereby creating a strictly anaerobic environment for the fermentation of Clostridium butyricum.

As a further improvement, in the final fermentation broth of the combined fermentation, the number of live bacteria and spores of Clostridium butyricum is not less than 2×10 ⁹ cfu/mL.

The beneficial effect of the above technical solution is that the present invention can significantly increase the amount of Clostridium butyricum by staggered inoculation of cerevisiae, Lactobacillus acidophilus and Clostridium butyricum, and the number of live bacteria and spores of Clostridium butyricum in the final combined fermentation liquid can reach 2.73×10 ⁹ cfu/mL and 2.65×10 ⁹ cfu/mL.

As a further improvement, in the final fermentation broth of the combined fermentation, the number of live bacteria of Lactobacillus acidophilus is not less than 1×10 ¹⁰ cfu/mL, and the number of live bacteria of Saccharomyces cerevisiae is not less than 3×10 ⁹ cfu/mL.

The beneficial effect of the above technical solution is that the invention utilizes staggered inoculation of saccharomyces cerevisiae, Lactobacillus acidophilus and Clostridium butyricum to maximize the rational utilization of resources, and the live counts of saccharomyces cerevisiae and Lactobacillus acidophilus in the final combined fermentation liquid can reach 3.71×10 ⁹ cfu/mL and 1.69×10 ¹⁰ cfu/mL.

As a further improvement, the components of the liquid culture medium include: 5-25 g/L glucose, 6-10 g/L bran, 5-15 g/L yeast extract, 5-20 g/L peptone, 0.1-5 g/L magnesium sulfate, 0.1-5 g/L dipotassium hydrogen phosphate, and 5-10 g/L calcium carbonate.

The beneficial effect of the above technical solution is that the present invention optimizes the components of the combined liquid culture medium based on the fermentation characteristics and production costs of Saccharomyces cerevisiae, Lactobacillus acidophilus and Clostridium butyricum. The optimized formula can ensure the smooth progress of the mixed fermentation process, obtain a high number of viable bacteria, and have a low cost.

As a further improvement, the pH control range during the combined liquid fermentation is 6.0-7.0.

The beneficial effect of the above technical solution is that controlling the pH between 6.0 and 7.0 during the combined liquid fermentation process can effectively increase the spore rate of Clostridium butyricum and can also increase the number of live bacteria of Clostridium butyricum, Lactobacillus acidophilus and Saccharomyces cerevisiae.

As a further improvement, the liquid filling coefficient of the fermentation tank during the combined liquid fermentation is 30% to 70%.

The beneficial effect of the above technical solution is that it saves production costs and reduces investment while ensuring successful fermentation.

As a further improvement, the inoculation amount of Clostridium butyricum, Lactobacillus acidophilus and Saccharomyces cerevisiae is 2% to 6%.

The beneficial effect of the above technical solution is that the inoculation amount can ensure the normal fermentation process, and the number of Clostridium butyricum and spores in the final product can reach a relatively high level.

As a further improvement, feeding was started 5-7 hours after inoculation of Clostridium butyricum and continued until the end of the logarithmic phase.

The beneficial effect of the above technical solution is that the timing and duration of feeding affect the final result of fermentation, and the spore rate of Clostridium butyricum in mixed culture can be effectively improved according to the above feeding scheme.

As a further improvement, the feeding includes a first feeding and a second feeding, and the second feeding starts directly after the first feeding ends; the feeding liquid composition of the first feeding is 4-10 g/L glucose, which lasts for 6-8 hours; the feeding liquid composition of the second feeding is 4-10 g/L glucose, 2-6 g/L peptone, and 1-3 g/L dipotassium hydrogen phosphate, which lasts until the end of the logarithmic phase. Specifically, the solvent of the feeding liquid is water.

The beneficial effect of the above technical solution is that the feeding is carried out in batches according to the composition of the feeding liquid, which can effectively ensure the maximization of energy utilization.

As a further improvement, the combined fermentation method includes inoculating the fermentation centrifuge liquid of the combined fermentation into a solid-state fermentation medium to obtain the feed additive through solid-state fermentation.

The beneficial effect of the above technical scheme is that the fermentation centrifugal waste liquid of the combined fermentation contains rich nutrients such as protein, polysaccharide and inorganic salt, and also contains bacteria that have not been completely centrifuged. After recycling it, it reduces the waste of resources caused by wastewater treatment, reduces the pressure on environmental protection, maximizes the use of resources, and reduces the cost of fermentation wastewater treatment for related enterprises.

As a further improvement, the components of the solid fermentation medium include 10% to 20% bran, 40% to 80% soybean meal, 1% to 10% wheat straw, 1% to 10% corn straw, 1% to 10% peanut straw, 0.05% to 0.15% magnesium sulfate, 0.5% to 1.5% calcium carbonate, and 0.5% to 1.5% dipotassium hydrogen phosphate.

The beneficial effect of the above technical solution is that the solid-state fermentation culture medium contains a large amount of straw, which recycles the agricultural by-product straw, saves resources, and reduces production costs for related companies.

As a further improvement, the solid-state fermentation condition is culturing at 30-37° C. for 3-7 days.

As a further improvement, the inoculation amount of the fermentation centrifuge liquid is 2% to 6% (v/m).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a growth curve of Clostridium butyricum, Lactobacillus acidophilus and Saccharomyces cerevisiae in Example 1 of the present invention;

FIG2 is the effect of carbon source on strain growth in Example 2 of the present invention;

FIG3 is the effect of nitrogen source on strain growth in Example 2 of the present invention;

FIG4 is the effect of inorganic salts on strain growth in Example 2 of the present invention;

FIG5 is the effect of temperature on strain growth in Example 3 of the present invention;

FIG6 is the effect of pH on strain growth in Example 3 of the present invention;

FIG7 is the effect of the liquid filling coefficient on the growth of the strain in Example 3 of the present invention;

FIG8 is a growth curve of each strain after optimization of shake flask culture conditions in Example 3 of the present invention;

9 is the effect of the carbon-nitrogen ratio of the feed solution on the growth of the strain during the fermentation process in a 10L tank in Example 4 of the present invention;

In Figure 1-9: the left vertical axis is applicable to the bacterial concentrations of Clostridium butyricum and Saccharomyces cerevisiae, and the right vertical axis is applicable to the bacterial concentration of Lactobacillus acidophilus.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with specific implementation methods, but the protection scope of the present invention is not limited thereto; unless otherwise specified, various reagents, instruments, etc. used in the examples are commercially available products.

Some of the biological materials, experimental reagents, experimental equipment, etc. involved in the following embodiments and test examples are briefly introduced as follows:

Culture medium:

The components of Clostridium butyricum activation medium are the same as those of seed medium, including: peptone 10 g/L, beef extract 10 g/L, yeast powder 3 g/L, glucose 5 g/L, soluble starch 1 g/L, sodium chloride 5 g/L, sodium acetate 3 g/L, L-cysteine hydrochloride 0.5 g/L, agar 0.5 g/L, pH 6.8;

Clostridium butyricum counting medium: add 2.0% agar powder to the RCM liquid medium;

The components of the Lactobacillus acidophilus activation medium and the seed medium are the same, both of which are MRS liquid medium, including: casein peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, sodium acetate 5g/L, diammonium citrate 2g/L, Tween 80 1g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate heptahydrate 0.2g/L, manganese sulfate heptahydrate 0.05g/L, calcium carbonate 20g/L, pH 6.8;

Lactobacillus acidophilus counting medium: add 2.0% agar powder to the MRS liquid medium;

The components of the activation medium for Saccharomyces cerevisiae and the seed medium are the same, both of which are YPD medium, including: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, pH 5.6;

Saccharomyces cerevisiae counting medium: Add 2.0% agar powder to the YPD liquid medium.

1. Specific embodiment of a combined fermentation method of Clostridium butyricum

Example 1 Determination of bacterial growth curve

Cultivation method of Clostridium butyricum: liquid deep static culture, conical flask filling coefficient 60%, inoculation amount 4%, sealed with 8 layers of gauze and 2 layers of kraft paper, static culture at 37℃ for 48 hours.

Cultivation method of Lactobacillus acidophilus: liquid deep static culture, conical flask filling coefficient 40%, inoculation amount 4%, sealed with 8 layers of gauze, static culture at 37℃ for 48h.

Cultivation method of brewer's yeast: liquid shake flask culture, conical flask filling coefficient 40%, inoculation amount 5%, culture at 30℃180r/min for 48h.

The bacteria were activated and cultured according to the above method. During the culture period, samples were taken every 4 hours to determine the number of live bacteria and their growth curves were drawn, as shown in Figure 1.

According to Figure 1, the growth curves of the three bacterial species show that the bacterial concentration is high in the late logarithmic period and the bacterial vitality is vigorous, so the seed culture time of Clostridium butyricum, Lactobacillus acidophilus and Saccharomyces cerevisiae is selected as 16h, 18h and 36h, respectively.

Example 2 Optimization of the components of the combined liquid culture medium in mixed fermentation

Initial fermentation method of mixed culture: According to the growth curves of Clostridium butyricum, Lactobacillus acidophilus and Saccharomyces cerevisiae, RCM medium was used as the initial combined culture medium. Saccharomyces cerevisiae was first inoculated into the combined liquid culture medium at a rate of 4%. After 6 hours, Lactobacillus acidophilus was inoculated into the combined liquid culture medium at a rate of 4%. After 14 hours, Clostridium butyricum was inoculated into the combined culture medium at a rate of 4%. The filling coefficient of the conical flask was 50%. The culture was statically incubated at 37°C and the conical flask was sealed with 8 layers of gauze and 2 layers of kraft paper.

The principle for selecting the time interval for staggered inoculation is as follows: According to the growth curves of Clostridium butyricum, Lactobacillus acidophilus and Saccharomyces cerevisiae in Figure 1, the time when the three strains enter the logarithmic growth phase, if Saccharomyces cerevisiae enters the logarithmic growth phase, it will grow rapidly and consume oxygen. This is the best time to inoculate Lactobacillus acidophilus, which has a lower oxygen demand. If the inoculation is late, more nutrients will be consumed, and the remaining nutrients for the target Clostridium butyricum will be reduced.

Based on the initial culture medium, the carbon source glucose was optimized, and 8 carbon sources including glucose, sucrose, lactose, maltose, soluble starch, corn starch, bran and soybean meal were selected to explore the effects of different carbon sources on the growth of co-cultured strains.

The selected optimized carbon source is used to select the preferred nitrogen source from peptone, tryptone, yeast extract, corn steep liquor powder, soybean meal powder, fish meal, beef extract and yeast extract powder.

According to the optimization results of the carbon source and nitrogen source types, an orthogonal experiment was designed to optimize the concentrations of the carbon source and nitrogen source. The carbon source concentration was set at 15g/L, 20g/L, and 25g/L, and the nitrogen source concentration was set at 10g/L, 20g/L, and 30g/L.

In the initial test, the inorganic salts selected included magnesium sulfate, manganese sulfate, potassium dihydrogen phosphate, sodium acetate trihydrate, calcium carbonate, sodium chloride, and potassium chloride. Based on the optimization of inorganic salt types, an orthogonal experiment was designed to optimize the inorganic salt concentration. In the optimization process, the number of live Clostridium butyricum and its spores, the number of live Lactobacillus acidophilus, and the number of live Saccharomyces cerevisiae were selected as the investigation indicators, with Clostridium butyricum as the main investigation object, and each group of experiments was repeated 3 times.

2.1. Optimization of carbon source

As can be seen from Figure 2, when glucose is used as the carbon source, the number of live Clostridium butyricum is much higher than that of other carbon sources; when maltose is used as the carbon source, the number of live Lactobacillus acidophilus and Saccharomyces cerevisiae is the largest; when bran is used as the carbon source, the number of live Clostridium butyricum, Lactobacillus acidophilus and Saccharomyces cerevisiae are all ideal. In order to further optimize the carbon source, glucose, maltose, sucrose and lactose were compounded with bran to explore the effect of compound carbon sources on the growth of bacteria. The results are shown in Table 1.

Table 1 Optimization results of composite carbon sources

Note: Combination 1 is glucose 12g/L + bran 8g/L; Combination 2 is maltose 10g/L + bran 10g/L; Combination 3 is sucrose 12g/L + bran 8g/L; Combination 4 is lactose 10g/L + bran 10g/L; Control is glucose 20g/L.

As shown in Table 1, the effect of the composite carbon source is better than that of a single carbon source. It can be seen that the composite carbon source is more conducive to the joint culture of three bacteria. When the composite carbon source is glucose and bran, the number of live bacteria and spores of Clostridium butyricum reaches the maximum value. When the composite carbon source is maltose and bran, the number of live bacteria of Lactobacillus acidophilus and Saccharomyces cerevisiae reaches the maximum value, and the number of live bacteria and spores of Clostridium butyricum is slightly lower than that of combination 1. Taking all factors into consideration, glucose and bran are selected as the carbon sources for joint culture.

2.2 Optimization of nitrogen source

As shown in Figure 3, when the nitrogen source is yeast extract, the number of live bacteria and spores of Clostridium butyricum are the highest, reaching 2.36×10 ⁷ cfu/mL and 2.12×10 ⁷ cfu/mL respectively, and the number of live bacteria of Lactobacillus acidophilus is also much higher than that of other nitrogen sources. When the nitrogen source is peptone, the number of live bacteria of Saccharomyces cerevisiae reaches the maximum. It was found from the relevant literature that compound nitrogen sources can achieve better results, so the results of this experiment combined yeast extract with other nitrogen sources, and the results are shown in Table 2.

Table 2 Optimization results of composite nitrogen source

Note: The ratio of yeast extract to other nitrogen sources is 1:1, the nitrogen source content is 20g/L, and the control is 20g/L yeast extract.

As shown in Table 2, in the combination of yeast extract and peptone as the composite nitrogen source, the number of live bacteria and spores of Clostridium butyricum and the number of live bacteria of Lactobacillus acidophilus reached the maximum value; when the composite nitrogen source was yeast extract and fish meal, the number of live bacteria of Saccharomyces cerevisiae reached the maximum value. In the combination of yeast extract and other nitrogen sources as the composite nitrogen source, the number of live bacteria of the three bacteria increased to varying degrees compared with the use of yeast extract alone. Taking comprehensive considerations, the composite nitrogen source of yeast extract and peptone was selected.

2.3 Optimization of carbon source and nitrogen source concentration ratio

The types of the best carbon and nitrogen sources were determined through single-factor experiments. Their concentrations and ratios also had a great influence on bacterial growth. Therefore, an orthogonal experiment was designed to further determine the optimal addition ratio of the composite carbon and nitrogen sources. The L9 (3 ⁴ ) orthogonal table was designed using SPSS 20.0 software as shown in Table 3.

Table 3 Orthogonal test factor level table of carbon source and nitrogen source concentration ratio

The results were analyzed by range and K value, and the results are shown in Table 4.

Table 4 Orthogonal test results of carbon source and nitrogen source concentration ratio

Note: A ₁ , B ₁ , and C ₁ correspond to the first level of each substance in the table, such as A ₁ represents the addition amount of glucose is 15 g/L.

It can be seen from Table 4 that the effect size of the number of live Clostridium butyricum is D＞B＞C＞A, indicating that peptone has a greater effect on the number of live Clostridium butyricum; the effect size of the number of Clostridium butyricum spores is B＞D＞A＞C, indicating that bran has a greater effect on the number of Clostridium butyricum transspores; the effect size of the number of live Lactobacillus acidophilus is B＞C＞A＞D, indicating that bran has a greater effect on the number of live Lactobacillus acidophilus; the effect size of the number of live Saccharomyces cerevisiae is C＞B＞D＞A, indicating that yeast extract has a greater effect on the number of live Saccharomyces cerevisiae. By comparing the K values, the optimized experimental conditions are: A ₃ B ₂ C ₃ D ₃ , but this method condition was not in the implemented experiment. The verification test results were: the live count of Clostridium butyricum was 4.67×10 ⁸ cfu/mL, the spore count of Clostridium butyricum was 3.63×10 ⁸ cfu/mL, the live count of Lactobacillus acidophilus was 9.35×10 ⁹ cfu/mL, and the live count of Saccharomyces cerevisiae was 2.27×10 ⁸ cfu/mL.

2.4. Optimization of inorganic salts

Although the effect of inorganic salts on fermentation bacteria is not as good as that of carbon and nitrogen sources, the addition of appropriate amounts can promote the healthy and rapid growth and reproduction of bacteria and increase the amount of their metabolites. The results of the effects of several common inorganic salts on bacterial growth are shown in Figure 4. The addition amounts of sodium chloride and potassium chloride are 5g/L, respectively, the addition amounts of dipotassium hydrogen phosphate and calcium carbonate are 1g/L and 5g/L, respectively, the addition amounts of manganese sulfate and magnesium sulfate are 0.3g/L, respectively, and the addition amount of sodium acetate trihydrate is 3g/L. The control is no addition of inorganic salts.

As shown in Figure 4, potassium hydrogen phosphate can promote the growth and transsporeization of Clostridium butyricum, and the viable counts of Lactobacillus acidophilus and Saccharomyces cerevisiae reached the maximum when the inorganic salt was calcium carbonate. In order to further determine the composition of the inorganic salt, potassium hydrogen phosphate and sodium acetate trihydrate, manganese sulfate, magnesium sulfate, and calcium carbonate, four inorganic salts with good results, were selected for optimization of the composite inorganic salt, and the results are shown in Table 5.

Table 5 Optimization results of composite inorganic salts

Note: Combination 1 is dipotassium hydrogen phosphate and sodium acetate trihydrate; combination 2 is dipotassium hydrogen phosphate and manganese sulfate; combination 3 is dipotassium hydrogen phosphate and magnesium sulfate; combination 4 is dipotassium hydrogen phosphate and calcium carbonate; the control is the addition of dipotassium hydrogen phosphate alone.

As shown in Table 5, the number of live bacteria of Clostridium butyricum in combination 3 and combination 4 is similar, and the number of spores of Clostridium butyricum in the combination of dipotassium hydrogen phosphate and calcium carbonate is better, at which the number of live bacteria of Clostridium butyricum reaches 4.05×10 ⁸ cfu/mL and the number of spores reaches 3.75×10 ⁸ . From this result, it can be seen that dipotassium hydrogen phosphate, magnesium sulfate and calcium carbonate can be selected as the best inorganic salt components in the combined culture medium.

The above experimental results show that potassium dihydrogen phosphate, magnesium sulfate and calcium carbonate are the best inorganic salts. SPSS 20.0 software was used to design the L9 (3 ³ ) orthogonal table to explore the concentration and ratio of the three, and the results were analyzed. The orthogonal test factors and levels are shown in Table 6.

Table 6 Factor level table of orthogonal test of compound inorganic salt ratio

The results were analyzed by range and K value, and the results are shown in Table 7.

Table 7 Results of orthogonal test on compound inorganic salt ratio

Note: A ₁ , B ₁ , and C ₁ correspond to the first level of each substance in the table, such as A ₁ represents the addition amount of dipotassium hydrogen phosphate is 2 g/L.

The range analysis of the experiment showed that the influence of live Clostridium butyricum was C>B>A; the influence of spore count was C>B>A; the influence of live Lactobacillus acidophilus was C>A>B; and the influence of live Saccharomyces cerevisiae was A>B>C. By comparing the K values, the optimal experimental conditions were A ₂ B ₃ C ₃ , that is, the addition of potassium hydrogen phosphate was 3.0 g/L, the addition of magnesium sulfate was 0.8 g/L, and the addition of calcium carbonate was 8 g/L. The live number of Clostridium butyricum was 5.24×10 ⁸ cfu/mL, the number of spores was 4.32×10 ⁸ cfu/mL, the live number of Lactobacillus acidophilus was 1.05×10 ¹⁰ cfu/mL, and the live number of Saccharomyces cerevisiae was 2.74×10 ⁸ cfu/mL.

From the above results of optimization of the components of the combined liquid culture medium in mixed fermentation, it can be seen that the optimal combined liquid culture medium of the present invention includes 25 g/L glucose, 6 g/L bran, 15 g/L peptone, 15 g/L yeast extract, 3 g/L dipotassium hydrogen phosphate, 0.8 g/L magnesium sulfate, and 8 g/L calcium carbonate.

Example 3 Optimization of combined fermentation culture conditions

Based on the optimal fermentation medium obtained in Example 2, the combined fermentation culture conditions were optimized. A 250 mL conical flask was filled with 50% volume of culture medium, with an initial pH of 6.5; sterilized, cooled, inoculated with 4% Clostridium butyricum, 4% Lactobacillus acidophilus, and 4% Saccharomyces cerevisiae seeds, and cultured at 37°C for 42 hours before detecting the bacterial concentration.

3.1. Optimization of fermentation temperature

The fermentation temperature was selected at 30℃, 33℃, 37℃, 40℃, 42℃, and 45℃ to investigate the effect of fermentation temperature on fermentation. The results are shown in Figure 5. As can be seen from Figure 5, the temperature has a significant effect on the growth of the strains during the combined fermentation process. When the temperature is in the range of 30-37℃, the concentration of Clostridium butyricum, Lactobacillus acidophilus, and Saccharomyces cerevisiae gradually increases with the increase of temperature. At 33℃, the number of live bacteria of Saccharomyces cerevisiae reaches a maximum value of 2.45×10 ⁸ cfu/mL. At 37℃, the number of live bacteria of Clostridium butyricum and Lactobacillus acidophilus both reached a maximum value of 4.96×10 ⁸ cfu/mL and 9.14×10 ⁹ cfu/mL, respectively. After the temperature exceeded 42℃, the number of live bacteria of Clostridium butyricum decreased significantly. Combined with the relevant literature and the results of this experimental study, the growth characteristics of the three bacteria should be comprehensively considered during the combined fermentation, so the temperature of the combined fermentation of the three bacteria was set at 37℃.

3.2. Optimization of pH

The pH in the combined fermentation culture was adjusted to 5.5, 5.7, 6.0, 6.5, 6.8, 7.2, and 7.5, respectively, and the effect of pH on the growth of strains during the combined fermentation process was investigated. The results in Figure 6 show that when the initial pH was 5.5, Clostridium butyricum hardly grew, and when the initial pH was in the range of 5.5 to 6.8, the number of live bacteria of Clostridium butyricum gradually increased with the increase of the initial pH, and when the initial pH was 6.8, the number of live bacteria of Clostridium butyricum reached the highest. Lactobacillus acidophilus can grow when the initial pH is 5.5, and the number of live bacteria reaches the maximum when the initial pH is 5.7. The growth curve of Saccharomyces cerevisiae with the change of pH is relatively gentle, and when the initial pH is 6.5, the number of live bacteria of Saccharomyces cerevisiae reaches the maximum. After consulting relevant literature and combining the research results of this experiment, the optimal initial pH for the combined fermentation of three bacteria is set to 6.8.

3.3 Optimization of liquid filling coefficient

Five gradients of 30%, 40%, 50%, 60% and 70% were selected to investigate the effect of the liquid filling coefficient on the combined fermentation. As shown in Figure 7, when the liquid filling coefficient was 50%, the live counts of Clostridium butyricum and Lactobacillus acidophilus reached the highest, and when the liquid filling coefficient was 40%, the live count of Saccharomyces cerevisiae reached the highest, but the live count of Clostridium butyricum decreased slightly. Considering that too high a liquid filling coefficient is not conducive to the growth of Clostridium butyricum, the optimal liquid filling coefficient for the combined fermentation of the three bacteria was set to 50%.

3.4. Optimization of inoculum size

The inoculation amounts of Clostridium butyricum, Lactobacillus acidophilus and Saccharomyces cerevisiae were set as three factors, each factor was set at 3 levels, and the experiment was conducted according to the L9 (3 ³ ) orthogonal table as shown in Table 8.

Table 8 Inoculation amount orthogonal test factor level table

The orthogonal test results were analyzed using SPSS 20.0 software, and the optimization results of the best inoculation ratio are shown in Table 9.

Table 9 Inoculum size orthogonal test results

Note: A ₁ , B ₁ , and C ₁ correspond to the first level of each substance in the table, such as A ₁ represents that the inoculation amount of Clostridium butyricum is 2%.

From the range analysis, it can be seen that the influence on the number of live Clostridium butyricum is C>A>B; the influence on the number of spores of Clostridium butyricum is A>C>B; the influence on the number of live Lactobacillus acidophilus is A>B>C; the influence on the number of live Saccharomyces cerevisiae is A>C>B. By comparing the K value, the optimal experimental conditions are A ₃ B ₁ C ₂ , that is, the inoculation amount of Clostridium butyricum is 5%, the inoculation amount of Lactobacillus acidophilus is 2%, and the inoculation amount of Saccharomyces cerevisiae is 4%. The results of the experiment with this inoculation are that the number of live Clostridium butyricum is 5.12×10 ⁸ cfu/mL, the number of spores is 4.23×10 ⁸ cfu/mL, the number of live Lactobacillus acidophilus is 1.01×10 ¹⁰ cfu/mL, and the number of live Saccharomyces cerevisiae is 2.41×10 ⁸ cfu/mL.

3.5. Optimization of fermentation time

According to the above fermentation method, first, brewer's yeast is inoculated into the combined liquid culture medium with an inoculation amount of 4% and cultured for 6 hours, then lactobacillus acidophilus is inoculated into the combined liquid culture medium with an inoculation amount of 2%, and after 14 hours, clostridium butyricum is inoculated into the combined culture medium with an inoculation amount of 5%. Samples are taken every 4 hours within 0 to 48 hours to detect the bacterial concentration to determine the culture time, and the results are shown in Figure 8. As can be seen from Figure 8, at 4 to 20 hours, brewer's yeast rapidly reproduces using the nutrients in the culture medium, and at 8 to 20 hours, lactobacillus acidophilus grows rapidly. The two bacteria consume the residual oxygen in the culture medium while multiplying in large quantities to provide an anaerobic environment for the growth of clostridium butyricum. At 18 hours, clostridium butyricum begins to reproduce and quickly enters the logarithmic phase. At 20 to 30 hours, clostridium butyricum grows together with lactobacillus acidophilus and brewer's yeast. Lactobacillus acidophilus and brewer's yeast provide a nutrient environment for the growth of clostridium butyricum, and clostridium butyricum provides growth factors such as vitamins for the other two bacteria. Clostridium butyricum began to produce spores at 34h, and the number of live bacteria reached the maximum at 42h, but the absolute value of the spore rate was slightly lower than that at 40h. Taking all factors into consideration, 42h was taken as the end point of mixed fermentation.

Example 4 Optimization of feeding method

A 10L fermenter was filled with 50% of the optimized combined liquid culture medium, the initial pH was adjusted to 6.8, sterilized at 121℃ for 30min, and the dissolved oxygen was set to 100% after cooling. 4% of Saccharomyces cerevisiae seeds were inoculated. After 6h of culture at 33℃ and 160rpm, when the dissolved oxygen dropped to 60%, 2% of Lactobacillus acidophilus was inoculated and cultured at 37℃. After 8h, the dissolved oxygen dropped to 10%, and 5% of Clostridium butyricum was inoculated and cultured at 37℃. During the static culture, the mixture was stirred at 50rpm for 2min every 20min to prevent the culture from precipitating. 10g/L glucose was added at 20h, and the flow was continued for 8h. After the first feeding, the second feeding was carried out immediately. The feeding liquid was a composite feeding liquid composed of 8g/L glucose, 2g/L peptone, and 1g/L potassium dihydrogen phosphate. The feeding was carried out until the end of the logarithmic phase, and the content and proportion of carbon source and nitrogen source of the composite feeding liquid were investigated. During the fermentation process, samples were taken regularly until 48h, and the bacterial concentration and residual sugar content of the fermentation liquid were detected. The results are shown in Figure 9. As can be seen from Figure 9, the carbon source in the feed solution has a greater impact on Saccharomyces cerevisiae, and the nitrogen source has a greater impact on Lactobacillus acidophilus. When there is only glucose in the feed solution, the number of live Clostridium butyricum bacteria increases. After adding nitrogen sources in different proportions, the number of live Clostridium butyricum bacteria and spore rate increase. When the carbon-nitrogen ratio is 2:1, the number of live Clostridium butyricum bacteria reaches the highest, the spore rate of Clostridium butyricum is also high, and the number of live bacteria in the fermentation liquid is also the highest. Taking all factors into consideration, the carbon-nitrogen ratio of the feed solution in the combined fermentation of Clostridium butyricum, Lactobacillus acidophilus and Saccharomyces cerevisiae is set to 2:1.

During the fermentation process, the pH was controlled at 6.8 or not adjusted, and the fermentation process was fed or not fed. The effects of pH and feeding on the fermentation were investigated. The specific results are shown in Table 10.

Table 10 Feed addition and pH adjustment fermentation results

Note: Control 1 was pH 6.8 without feeding, and Control 2 was natural fermentation without feeding and pH adjustment.

As shown in Table 10, feed supplement and pH control have an important influence on the fermentation results. Feed supplement and pH control can effectively increase the spore rate of Clostridium butyricum, and can also increase the number of live bacteria of Clostridium butyricum, Lactobacillus acidophilus and Saccharomyces cerevisiae.

Example 5 Recycling of combined fermentation centrifugal wastewater

The combined fermentation liquid was centrifuged at 4000 rpm for 10 min, and the bacterial sludge was used to prepare bacterial powder by spray drying. The supernatant contained 1.25×10 ⁷ cfu/mL of Clostridium butyricum, 2.76×10 ⁸ cfu/mL of Lactobacillus acidophilus, and 2.54×10 ⁶ cfu/mL of Saccharomyces cerevisiae, and could be used as seed liquid for solid feed fermentation.

Preparation of solid culture medium: First, crush peanuts, corn and wheat straw to 50 mesh. Then, mix peanut straw, corn straw, wheat straw, bran, soybean meal, magnesium sulfate, calcium carbonate and dipotassium hydrogen phosphate according to the content designed in this embodiment, and sterilize at 121° C. for 20 minutes.

Take 5.0 kg of sterilized solid fermentation medium and put it into a fermentation bag with a one-way air valve, 500 g per bag. Add the supernatant into the bag in different proportions, stir evenly and seal the bag. Place it in an incubator and culture it at 37°C for 7 days. During this period, shake the material every day to mix it evenly.

The bran-soybean meal ratio, feed-water ratio and bacterial inoculation amount in the solid-state fermentation medium are relatively important influencing factors in the solid-state feed fermentation process. Based on the literature and the experience accumulated in this project, the following process optimization research was carried out.

5.1. Optimization of bran-soybean meal ratio, feed-water ratio and supernatant inoculum amount

In order to achieve the best solid-state fermentation results, the ratio of crushed peanut straw, corn straw and wheat straw was 1:1:1 (each accounting for 6.5%), the total amount of bran and soybean meal was 80%, the addition of magnesium sulfate was 0.1%, the addition of dipotassium hydrogen phosphate was 0.1%, and the addition of calcium carbonate was 0.5% as the initial conditions. The bran and soybean meal ratio, feed-water ratio and supernatant inoculation amount were selected as three factors, and the orthogonal test was carried out according to the L9 (3 ³ ) orthogonal table as shown in Table 11.

Table 11 Orthogonal test factor levels of bran soybean meal ratio, feed-water ratio and supernatant inoculation amount

The orthogonal test results were analyzed using SPSS 20.0 software, and the best optimization results are shown in Table 12.

Table 12 Orthogonal test results of bran soybean meal ratio, feed-water ratio and supernatant inoculation amount

Note: A ₁ , B ₁ , and C ₁ correspond to the first level of each substance in the table, such as A ₁ represents a bran-soybean meal ratio of 1:2.

The analysis of the range showed that the influence of each factor on the number of live Clostridium butyricum was A>C>B, the influence on the number of live Lactobacillus acidophilus was C>A>B, and the influence on the number of live Saccharomyces cerevisiae was C>B>A. By comparing the K value, the optimal experimental conditions were A ₂ B ₁ C ₂ , that is, the bran soybean meal ratio was 1:3, the feed-water ratio was 1:0.5, and the inoculation amount of the supernatant was 15%. The verification test was carried out under these conditions, and the results were: the number of live Clostridium butyricum was 3.93×10 ⁷ cfu/g, the number of live Lactobacillus acidophilus was 7.51×10 ⁸ cfu/g, and the number of live Saccharomyces cerevisiae was 7.94×10 ⁶ cfu/g.

5.2. Optimization of the addition ratio of wheat, corn and peanut straw

After obtaining the above fermentation conditions, on the basis of these conditions, three factors, namely the proportion of wheat straw addition, corn straw addition and peanut straw addition, were selected, and an orthogonal test was carried out according to the L9 (3 ³ ) orthogonal table as shown in Table 13.

Table 13 Factor level table of orthogonal test of wheat, corn and peanut straw addition ratio

The orthogonal test results were analyzed using SPSS 20.0 software, and the best optimization results are shown in Table 14.

Table 14 Results of orthogonal test on the addition ratio of wheat, corn and peanut straw

Note: A ₁ , B ₁ , and C ₁ correspond to the first level of each substance in the table, such as A ₁ represents that the addition ratio of wheat straw is 15%.

The analysis of the range showed that the influence of each factor on the number of live Clostridium butyricum was B>C>A, the influence on the number of live Lactobacillus acidophilus was C>B>A, and the influence on the number of live Saccharomyces cerevisiae was A>B>C. By comparing the K value, the optimal addition ratio of wheat straw, corn straw, and peanut straw was A ₁ B ₂ C ₃ , that is, the addition ratio of wheat straw was 5%, and the addition ratios of corn straw and peanut straw were both 7%. Under this condition, the verification test was carried out, and the results were: the number of live Clostridium butyricum was 2.63×10 ⁸ cfu/g, the number of live Lactobacillus acidophilus was 4.62×10 ⁹ cfu/g, and the number of live Saccharomyces cerevisiae was 2.56×10 ⁸ cfu/g.

5.3. Optimization of the amount of inorganic salt added

In order to further optimize the addition amount of inorganic salts, on the basis of the above optimization conditions, three factors of magnesium sulfate, dipotassium hydrogen phosphate and calcium carbonate addition amount were selected, and an orthogonal test was carried out according to the L9 (3 ³ ) orthogonal table as shown in Table 15.

Table 15 Inorganic salt addition orthogonal test factor level table

The orthogonal test results were analyzed using SPSS 20.0 software, and the best optimization results are shown in Table 16.

Table 16 Results of orthogonal test on inorganic salt addition

Note: A ₁ , B ₁ , and C ₁ correspond to the first level of each substance in the table, such as A ₁ represents the addition amount of magnesium sulfate is 0.1 g/L.

Analysis of the range showed that the influence of each factor on the number of live Clostridium butyricum was A＞B＞C, the influence on the number of live Lactobacillus acidophilus was C＞B＞A, and the influence on the number of live Saccharomyces cerevisiae was B＞A＞C. By comparing the K value, the optimal addition ratio of magnesium sulfate, dipotassium hydrogen phosphate and calcium carbonate was A ₁ B ₂ C ₃ , that is, the addition amount of magnesium sulfate was 0.1g/L, the addition amount of dipotassium hydrogen phosphate was 0.3g/L, and the addition amount of calcium carbonate was 10g/L. Under this condition, the verification test was carried out, and the results were as follows: the number of live Clostridium butyricum was 4.86×10 ⁸ cfu/g, the number of live Lactobacillus acidophilus was 6.65×10 ⁹ cfu/g, and the number of live Saccharomyces cerevisiae was 4.57×10 ⁸ cfu/g.

In summary, the orthogonal test above has obtained the optimal experimental conditions for solid-state fermentation using centrifugal supernatant and crop by-products, that is, the bran soybean meal ratio is 1:3 (bran 20%, soybean meal 60%), the addition ratio of wheat straw is 5%, the addition ratio of corn straw and peanut straw is 7%, the addition amount of magnesium sulfate is 0.1%, the addition amount of potassium hydrogen phosphate is 0.3%, and the addition amount of calcium carbonate is 1%. The material-water ratio is 1:0.5, and the inoculation amount of the supernatant is 15%. After solid-state fermentation, the number of live bacteria of Clostridium butyricum in the solid-state fermentation product reached 4.86×10 ⁸ cfu/g, the number of live bacteria of Lactobacillus acidophilus reached 6.65×10 ⁹ cfu/g, and the number of live bacteria of Saccharomyces cerevisiae reached 4.57×10 ⁸ cfu/g.

2. Experimental Examples

Experimental Example 1 Comparison of liquid fermentation effects

In addition to exploring a combined liquid fermentation process of Clostridium butyricum, Lactobacillus acidophilus and Saccharomyces cerevisiae, a combined liquid fermentation method of Clostridium butyricum and Saccharomyces cerevisiae was also explored in the early stage of this application, and the process was optimized. The results are shown in Table 17.

Table 17 Comparison of fermentation results of different fermentation strains in combined liquid fermentation

As shown in Table 17, the addition of Lactobacillus acidophilus can further consume oxygen in the fermentation process, provide a good environment for the fermentation of Clostridium butyricum, and increase the number of live Clostridium butyricum and spores in the fermentation final product.

Experimental Example 2 Comparison of solid-state fermentation effects

Liquid fermentation was carried out according to the culture medium formula and fermentation process optimized in Examples 2 to 4, and the supernatant obtained by centrifugation of the obtained fermentation liquid was used as seeds for solid feed fermentation.

The supernatant was inoculated into the solid fermentation medium optimized in Example 5 at a ratio of 15%, and was inoculated into the traditional solid medium (including 24.7% bran, 74.1% soybean meal, 0.1% magnesium sulfate, 0.3% potassium dihydrogen phosphate, and 1% calcium carbonate) at a ratio of 15%, sealed with sealing film, and placed in an incubator at 37°C for 42 hours. The results are shown in Table 18.

Table 18 Comparison of solid-state fermentation effects

As can be seen from Table 18, compared with the traditional solid culture medium, the viable counts of Clostridium butyricum, Lactobacillus acidophilus and Saccharomyces cerevisiae obtained by solid fermentation using peanut straw, corn straw and wheat straw as part of the culture medium components are comparable to the traditional solid fermentation results. It can be seen that using straw to replace part of the culture medium components can also achieve relatively ideal fermentation results, but at a lower cost.

Experimental Example 3 Animal feeding experiment of solid-state fermentation products

This experimental example is used to illustrate the effects of solid-state fermentation products on the growth status and immunity of calves.

Thirty healthy calves of about 50 days old (80 kg) were selected and randomly divided into three groups, 10 in each group. The control group was fed a basal diet, and the experimental group one was fed with 0.5 g/Kg of the solid-state fermentation product in Example 5 according to the calf weight per day on the basis of the basal feed. The experimental group two was fed with 1 g/Kg of the composite bacterial agent in patent CN108048355A "A butyric Clostridium butyricum fermentation product and a butyric Clostridium butyricum solid-state fermentation method" according to the calf weight per day (prepared according to the preparation method recorded in the patent, the main components of the composite bacterial agent include soybean meal, wheat bran, corn flour, alkaline protease and α-amylase, etc.), and the calves were fed freely in a funnel-type feeding trough, with sufficient water supply and daily management as usual. A feeding experiment for a total of 35 days was carried out.

The basic feed ingredients are: corn 44.5%, soybean meal 20%, cottonseed meal 10%, bran 20%, stone powder 1%, calcium bicarbonate 1.5%, salt 0.8%, baking soda 1.2%, and premix 1%.

The feed was weighed at the beginning (0 day), 7 days, 21 days and the end (35 days) of the experiment, and the calves were weighed at the end (35 days). The number of calves with diarrhea was counted at 9 am every day, and the average daily gain (ADG), average daily feed intake (ADFI) and feed: gain ratio (F/G) were calculated.

Average daily gain (ADG) = (weight at the end of the test - weight at the beginning of the test) / number of days of the test;

Feed-to-weight ratio = average daily feed intake/average daily weight gain;

Diarrhea rate = number of diarrhea cattle/(number of test cattle × total number of days) × 100%,

Among them, the number of calves with diarrhea during the experimental period = the number of calves with diarrhea on the first day + the number of calves with diarrhea on the second day + ... the number of calves with diarrhea on the 35th day.

SAS (Statistical Analysis) version 7.2 was used for statistical analysis and variance analysis, and the results were considered significant when P < 0.05. Significant differences were expressed by letter marking: first, all the averages were arranged in order from large to small, and then the letter a was marked on the largest average; and the average was compared with the following averages, and all those with insignificant differences were marked with the letter a, until a certain average with significant difference was marked with the letter b; then the average marked with b was used as the standard, and compared with the averages above that were larger than it, and all those with insignificant differences were also marked with the letter b; then the largest average marked with b was used as the standard, and compared with the following unmarked averages, all those with insignificant differences continued to be marked with the letter b, until a certain average with significant difference was marked with c. The results are shown in Table 19, and the average value of each group of data was taken.

Table 19 Comparison of animal feeding experimental effects

Group Starting weight/kg Final weight/kg Daily weight gain/g Material weight ratio Diarrhea rate Control group 80.1 ^a 91.4 ^a 0.32 ^a 6.5 ^a 14.2 ^a Experimental Group 1 79.8 ^a 94.1 ^b 0.41 ^c 4.5 ^b 9.8 ^c Experimental Group 2 79.9 ^a 93.8 ^b 0.38 ^c 5.2 ^b 11.1 ^b

Note: The letters a, b, and c on the shoulders of the numbers in the same column indicate extremely significant differences (P < 0.01), and the same letters indicate no significant differences (P > 0.05).

From the statistical analysis results of Table 19, it can be seen that the experimental group has significant differences from the control group in terms of average daily weight gain, feed-to-weight ratio, and diarrhea rate (P < 0.05). Experimental group one is significantly different from experimental group two in reducing the diarrhea rate of calves, and the amount of addition is only half of that of the experimental group. In addition, straw is used as a raw material in the solid-state fermentation of this patent, and the cost of solid-state fermentation products is lower. Therefore, from a comprehensive evaluation of feeding costs and growth performance, the product of solid-state fermentation of straw using the supernatant of the combined fermentation of Clostridium butyricum, Lactobacillus acidophilus and Saccharomyces cerevisiae can meet the needs of some calf breeding, and achieve the effect of improving calf growth performance and reducing diarrhea rates.

On the end of the experiment, the calves were fasted for 12 h. 5 mL of blood was collected from the jugular vein of 5 calves in each group. The blood was centrifuged at 3500 r/min for 10 min, and the upper serum was collected and stored at -20°C for the determination of immunoglobulin G (Ig G) content in calf serum.

The average IgG content in the serum of calves in experimental group one was 9.96 g/L, and the average IgG content in the serum of calves in experimental group two was 9.63 g/L, and there was a significant difference between experimental group one and experimental group two (P < 0.05). The average IgG content in the serum of calves in the control group was 7.84 g/L, and there was a significant difference between the experimental group and the control group (P < 0.05).

Based on the above results, it can be seen that the solid-state fermentation product using the centrifuge liquid of mixed fermentation can improve the growth performance of calves, increase the serum immune level, and reduce the diarrhea rate.

Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A combined fermentation method of clostridium butyricum, which is characterized by comprising the following steps: sequentially and timely inoculating Saccharomyces cerevisiae, lactobacillus acidophilus and Clostridium butyricum into a liquid culture medium, and performing joint fermentation of Clostridium butyricum without additionally providing an anaerobic environment.

2. The method for the combined fermentation of clostridium butyricum according to claim 1, wherein: the time-staggered inoculation is to inoculate Saccharomyces cerevisiae into a liquid culture medium, inoculate lactobacillus acidophilus after 5-8 hours and inoculate clostridium butyricum after 12-16 hours.

3. The method for the combined fermentation of clostridium butyricum according to claim 1, wherein: in the final fermentation broth of the combined fermentation, the viable count and the spore count of clostridium butyricum are not less than 2 multiplied by 10 ⁹ cfu/mL。

4. A combined fermentation process of clostridium butyricum according to claim 3 wherein: in the final fermentation broth of the combined fermentation, the viable count of lactobacillus acidophilus is not less than 1 multiplied by 10 ¹⁰ cfu/mL, the viable count of Saccharomyces cerevisiae is not less than 3×10 ⁹ cfu/mL。

5. The method for the combined fermentation of clostridium butyricum according to claim 1, wherein: the liquid culture medium comprises the following components: glucose 5-25 g/L, bran 6-10 g/L, yeast extract 5-15 g/L, peptone 5-20 g/L, magnesium sulfate 0.1-5 g/L, dipotassium hydrogen phosphate 0.1-5 g/L, and calcium carbonate 5-10 g/L.

6. The method for the combined fermentation of clostridium butyricum according to claim 1, wherein: and starting feeding 5-7 hours after inoculating clostridium butyricum, and finishing the feeding until the logarithmic phase is finished.

7. The method for the combined fermentation of clostridium butyricum according to claim 6, wherein: the material supplementing comprises a first material supplementing and a second material supplementing, and the second material supplementing is directly started after the first material supplementing is finished; the composition of the feed supplement liquid of the first feed supplement is glucose 4-10 g/L, and the time lasts for 6-8 hours; the feed liquid of the second feed is composed of 4-10 g/L glucose, 2-6 g/L peptone and 1-3 g/L dipotassium hydrogen phosphate, and the process is continued until the logarithmic phase is finished.

8. The method for the combined fermentation of clostridium butyricum according to claim 1, wherein: the combined fermentation method comprises the steps of inoculating fermentation centrifugate of combined fermentation into a solid fermentation medium, and obtaining the feed additive through solid fermentation.

9. The method for the combined fermentation of clostridium butyricum according to claim 8, wherein: the solid state fermentation culture medium comprises 10% -20% of bran, 40% -80% of soybean meal, 1% -10% of wheat straw, 1% -10% of corn straw, 1% -10% of peanut straw, 0.05% -0.15% of magnesium sulfate, 0.5% -1.5% of calcium carbonate and 0.5% -1.5% of dipotassium hydrogen phosphate.

10. The method for the combined fermentation of clostridium butyricum according to claim 8, wherein: the condition of the solid state fermentation is that the solid state fermentation is cultured for 3-7 d at 30-37 ℃.

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