CN111500508B - Liquid mixed fermentation method of clostridium butyricum and bacillus coagulans - Google Patents
Liquid mixed fermentation method of clostridium butyricum and bacillus coagulans Download PDFInfo
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Abstract
The invention relates to a mixed fermentation method of liquid of clostridium butyricum and bacillus coagulans, which specifically comprises the following steps: (1) preparing a fermentation medium, putting the fermentation medium into a fermentation tank, adjusting the initial pH of the fermentation medium to 6.0-7.5, and sterilizing at 121 ℃ for 30min, wherein the components of the fermentation medium comprise 0.5-20 g/L of bran, 0.5-25 g/L of peptone, 1-25 g/L of corn steep liquor dry powder, 0.1-5 g/L of dipotassium hydrogen phosphate and 0.1-5 g/L of manganese sulfate; (2) carrying out liquid culture on clostridium butyricum and bacillus coagulans strains to obtain inoculated liquid; (3) inoculating clostridium butyricum and bacillus coagulans into a fermentation tank, fermenting at 33-37 ℃, and standing for culture, wherein the total fermentation time is 32-44 h. The invention adopts a mixed fermentation mode, and the viable count of clostridium butyricum reaches 5.8 multiplied by 108cfu/mL, the spore rate is 99.4%; the viable count of the bacillus coagulans reaches 4.7 multiplied by 108cfu/mL, and provides a new energy-saving and environment-friendly fermentation mode for the industrial production of clostridium butyricum.
Description
Technical Field
The invention belongs to the technical field of microbial agent production processes, and particularly relates to a mixed fermentation method of liquid of clostridium butyricum and bacillus coagulans.
Background
A microecological preparation, also called as microecological regulator, is prepared from normal microorganisms beneficial to the host, metabolites thereof and growth promoting substances according to the microecological principle, and can regulate and maintain the microecological balance and improve the health state of the host through the actions of antibiosis, adhesion planting, biological barrier and the like. The physiological functions of the microecological preparation mainly comprise: immunoregulation effect, antibacterial effect, nutrition effect, anti-tumor effect, and blood lipid reducing effect. The microecological preparation comprises probiotics, prebiotics and synbiotics. Currently, most of the microecologics used in clinic are probiotic preparations. The probiotics commonly used in clinic comprise strains such as lactobacillus, clostridium butyricum (clostridium butyricum), bifidobacterium infantis, enterococcus faecium, streptococcus faecalis, bacillus subtilis, bacillus cereus, bacillus licheniformis, saccharomycetes and the like. In recent years, microecologics have attracted increasing attention and play an important role in the fields of agriculture, feed, food, medicine and health care.
The clostridium butyricum is a spore-producing probiotic which is popular in recent years, has strong stress resistance, can promote the proliferation of probiotics such as bifidobacterium, lactobacillus and the like, and has an inhibiting effect on various pathogenic bacteria such as escherichia coli, staphylococcus aureus and the like. In addition, the study shows that the clostridium butyricum can improve the immunity of the organism and also has the anticancer effect. Clostridium butyricum, on the other hand, is sensitive to only a few antibiotics and can therefore be used in combination with some antibiotics to reduce antibiotic abuse.
The clostridium butyricum is widely used for the auxiliary treatment of intestinal flora imbalance and low body immunity caused by various diseases in clinic. The clostridium butyricum preparation has wide application prospect in animal husbandry, and can improve the production performance of animals, enhance the resistance of the animals, improve the conversion rate of the feed, improve the laying rate, reduce the feed conversion ratio and save the feeding cost when being added into the feed.
Clostridium butyricum is a strict anaerobic bacterium, and nitrogen needs to be supplemented to the clostridium butyricum in the fermentation process to provide an absolute anaerobic environment, so that the complexity of equipment and the difficulty of the process are increased, and mixed fermentation is one of effective methods for solving the problem. In the mixed fermentation process, the characteristics of absolute anaerobism of clostridium butyricum and facultative anaerobism of bacillus coagulans are utilized, and oxygen in a culture medium is consumed through the growth of the bacillus coagulans, so that an anaerobic environment is created for the growth of the clostridium butyricum. In the prior art, the multi-bacterium mixed fermentation is solid state fermentation, and no report of liquid multi-bacterium mixed fermentation is found.
Disclosure of Invention
The invention provides a mixed fermentation method of clostridium butyricum and bacillus coagulans liquid, fills the blank that no mixed fermentation of clostridium butyricum and bacillus coagulans liquid exists in the prior art, and provides a new energy-saving and environment-friendly mode for a fermentation process of clostridium butyricum.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a liquid mixed fermentation method of Clostridium butyricum and Bacillus coagulans comprises the following steps:
(1) preparing a fermentation medium, filling the fermentation medium into a fermentation tank, adjusting the initial pH of the fermentation medium to 6.0-7.5, and sterilizing at 121 ℃ for 30 min;
(2) carrying out liquid culture on clostridium butyricum and bacillus coagulans strains to obtain inoculated liquid;
(3) inoculating clostridium butyricum and bacillus coagulans into a fermentation tank, fermenting at 33-37 ℃, and standing for culture, wherein the total fermentation time is 32-44 h.
Preferably: the fermentation medium comprises 0.5-20 g/L of bran, 0.5-25 g/L of peptone, 1-25 g/L of corn steep liquor dry powder, 0.1-5 g/L of dipotassium hydrogen phosphate and 0.1-5 g/L of manganese sulfate.
Preferably: the liquid filling coefficient of the fermentation tank is 20-60%.
Preferably: the inoculation amount of the clostridium butyricum is 2-6%, and the inoculation amount of the bacillus coagulans is 2-8%.
Preferably: the inoculation liquid of the clostridium butyricum is obtained by carrying out deep static culture on a liquid at 37 ℃ for 12-16 h, and the inoculation liquid of the bacillus coagulans is obtained by carrying out stirring culture at 37 ℃ for 16-18 h.
Preferably: and the components of the supplementary material liquid obtained by the second supplementary material are 0-5 g/L of glucose, 0-3.0 g/L of peptone and 0.10-0.50 g/L of dipotassium hydrogen phosphate.
Preferably: the fermentation method also comprises the step of adjusting the pH value to be 5.5-6.5 in the fermentation process.
Preferably: the fermentation method further comprises the steps of starting a fermentation tank for stirring, adjusting the rotation speed to be 20-150 rpm, and performing first material supplement, wherein the components of a supplemented liquid are 2-10 g/L of glucose and last for 8 hours; and after the first feeding, performing second feeding, wherein the components of the feeding liquid are a composite feeding liquid consisting of 1-6 g/L of glucose, 1-6 g/L of peptone and 0.1-1 g/L of dipotassium hydrogen phosphate, and feeding till the end of the logarithmic phase.
Has the advantages that:
according to relevant documents, the process research of clostridium butyricum mostly stays at the shake flask level, and anaerobic agents or nitrogen are required to be added to provide an anaerobic environment for the clostridium butyricum during the culture process. At present, strict anaerobic environment needs to be provided for domestic industrial production of clostridium butyricum. According to the research, the mixed fermentation is carried out according to different growth characteristics of the clostridium butyricum and the bacillus coagulans, so that the production cost is reduced, and the development requirement of energy conservation and environmental protection is met.
The research adopts a mixed fermentation mode, and the viable count of clostridium butyricum reaches 5.8 multiplied by 108
The spore rate of the clostridium butyricum is 99.4 percent in cfu/mL, and the viable count of the bacillus coagulans reaches 4.7 multiplied by 108cfu/mL, which provides a new fermentation mode for the industrial production of clostridium butyricum. Although there are reports of solid-state mixed fermentation, there are no reports of liquid-state mixed fermentation of Clostridium butyricum and Bacillus coagulans. The invention belongs to the initiative at home and abroad.
Drawings
In order to more clearly explain the embodiments of the present invention, drawings used in the embodiments are briefly described. It is to be understood that the drawings in the following description are only a part of the invention.
FIG. 1 shows the growth curve and pH change curve of Clostridium butyricum DL-1;
FIG. 2 shows the growth curve and pH variation curve of Bacillus coagulans;
FIG. 3 effect of carbon source on growth of strain;
FIG. 4 effect of nitrogen source on growth of strain;
FIG. 5 effect of inorganic salts on growth of the strains;
FIG. 6 effect of temperature on growth of the strain;
FIG. 7 effect of pH on strain growth;
FIG. 8 influence of liquid loading factor on growth of the strain;
FIG. 9 growth curve and pH variation curve of the strains in shake flask culture;
FIG. 1010L effect of feed liquid carbon nitrogen ratio on strain growth during tank fermentation.
Detailed Description
The embodiments of the present invention will be described in detail and fully with reference to the accompanying drawings. It is to be understood that the described embodiments are merely illustrative of some, but not all, of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The following examples refer to media
Bacillus coagulans slant culture medium: beef extract 5g/L, tryptone 10g/L, sodium chloride 5g/L, pH 7.0 and 0.1.
Bacillus coagulans seed culture medium: 10g/L of glucose, 10g/L of tryptone, 5g/L of yeast extract and 6.8-7.0 of pH.
Bacillus coagulans counting medium: 20g/L of bran, 10g/L of yeast extract, 20g/L of tryptone, 1000mL of distilled water, 6.8 of pH and 1.5% of agar powder.
Clostridium butyricum slant culture medium: 5g/L beef extract, 10g/L tryptone and 5g/L, pH 7.0.0 sodium chloride and 0.1.
Clostridium butyricum seed culture medium: 5g/L of glucose, 5g/L of sodium chloride, 3g/L of sodium acetate trihydrate, 10g/L of tryptone, 3g/L of yeast extract, 10g/L of beef extract, 1g/L, L of soluble starch-0.5 g/L of cysteine hydrochloride and 7.2-7.3.
Clostridium butyricum counting medium: tryptone 15g/L, peptone 5g/L, yeast extract 5g/L, ferric ammonium citrate 1g/L, sodium metabisulfite 1g/L, distilled water 1000mL, pH7.4, 0.1, 1.1% agar.
EXAMPLE 1 Strain growth Curve determination
The clostridium butyricum is cultured by a liquid deep layer standing method, the liquid filling coefficient of an erlenmeyer flask is 60 percent, the inoculation amount is 4 percent, 8 layers of gauze and 2 layers of kraft paper are used for sealing, and standing culture is carried out at 37 ℃.
The culture mode of the bacillus coagulans is as follows: the coefficient of the liquid contained in the Erlenmeyer flask is 20%, the inoculum size is 4%, and the Erlenmeyer flask is cultured at 37 ℃ and 180 rpm.
The strain activation and culture are carried out according to the method, the clostridium butyricum samples every 4 hours for 0 to 48 hours to determine the OD600 value, the bacillus coagulans samples every 2 hours for 0 to 24 hours to determine the OD600 value, the growth curves are respectively drawn, and the pH value of the bacterial liquid is determined at the same time. The growth curves and pH curves of Clostridium butyricum and Bacillus coagulans are shown in FIGS. 1 and 2.
As can be seen from FIG. 1, in the activated medium, the lag phase of Clostridium butyricum is short, and the cells begin to rapidly proliferate at the 4 th hour and enter the logarithmic phase, while the pH sharply decreases, and the cells reach the maximum concentration at the end of the logarithmic phase at 12h and reach the maximum concentration at 16h, and the pH at this time is also close to the minimum. After 20h, the thalli enters the decline period and the pH value is slightly increased. Therefore, 12-16 h is selected as the culture time of the clostridium butyricum seeds, and 16h is more preferable.
As can be seen from FIG. 2, the growth of the Bacillus coagulans in the activation medium is slow within 0-8 h, the pH value is also slowly reduced, the thalli are rapidly propagated from 8h and enter a logarithmic phase, the thalli enter a stationary phase within 16h, the concentration of the thalli reaches the highest value within 18h, and the pH value also reaches the lowest value at the moment. Therefore, 16h to 18h are selected as the culture time of the bacillus coagulans seeds, and 18h is more preferable. The following examples used 16h cultured Clostridium butyricum seeds and 18h cultured Bacillus coagulans seeds.
Example 2 Mixed fermentation Medium composition optimization
Mixed culture initial fermentation mode: the coefficient of the liquid contained in the erlenmeyer flask is 40%, the initial inoculation amount of clostridium butyricum is 6%, the initial inoculation amount of bacillus coagulans is 4%, the erlenmeyer flask is statically cultured at 37 ℃, and the erlenmeyer flask is sealed by 8 layers of gauze and 2 layers of kraft paper.
Glucose, sucrose, maltose, lactose, bran, soluble starch, corn starch and the like are selected as alternative carbon sources, and the concentration is 10 g/L. Tryptone 10g/L and yeast extract 5g/L as initial nitrogen source.
Selecting a preferable nitrogen source from corn steep liquor dry powder, soybean meal powder, fish meal, beef extract, peptone, tryptone, yeast extract and yeast extract powder by using a selected optimized carbon source, wherein the addition amount is 15 g/L.
Designing an orthogonal test according to the optimization results of the types of the carbon source and the nitrogen source, and optimizing the concentrations of the carbon source and the nitrogen source, wherein the concentrations of the carbon source are set to be 10g/L, 15g/L and 20g/L, and the concentrations of the nitrogen source are set to be 5g/L, 10g/L and 15 g/L.
According to the literature, the inorganic salts selected in the initial test include 5g/L of sodium chloride, 5g/L of potassium chloride, 0.3g/L of magnesium sulfate, 0.3g/L of manganese sulfate, 1g/L of dipotassium hydrogen phosphate, 3g/L of acetic acid trihydrate and 1g/L of calcium carbonate. On the basis of the optimization of the types of the inorganic salts, an orthogonal test is designed to optimize the concentration of the inorganic salts. And optimizing the obtained result by using the carbon source and the nitrogen source. The preliminary test shows that the bacillus coagulans basically does not produce spores during mixed fermentation, and the number of the viable bacteria is far lower than the result of single-bacterium fermentation, so that the number of the viable bacteria of the clostridium butyricum, the number of the spores of the clostridium butyricum and the number of the viable bacteria of the bacillus coagulans are selected as investigation indexes in the optimization process, and the clostridium butyricum is used as a main investigation object.
FIGS. 3 to 5 show the effect of the carbon source, nitrogen source and inorganic salt species on the mixed culture.
As can be seen from FIG. 3, the number of viable bacteria of Clostridium butyricum is much higher than that of other carbon sources when bran is used as the carbon source, and sucrose is used as the secondary carbon source; the spore number of clostridium butyricum is better than that of other carbon sources when bran is used as the carbon source; the carbon source has little influence on the viable count of the bacillus coagulans. A large number of studies show that when the culture medium contains starch, the growth of clostridium butyricum is facilitated, and the results of the experiment are combined, the bran and the starch are compounded, so that the carbon source is further optimized, and the results are shown in Table 1.
TABLE 1 composite carbon source optimization results
Note: combining one of 8g/L bran and 2g/L soluble starch as a composite carbon source, combining two of 5g/L bran and 5g/L soluble starch as a composite carbon source, and comparing with 10g/L bran.
As can be seen from Table 1, when the carbon source is bran only, the viable count of Clostridium butyricum is higher than that of the mixture of bran and soluble starch, and the viable count of Clostridium butyricum is greatly reduced when the content of the soluble starch is increased. Therefore, the addition of soluble starch is not beneficial to the growth of butyric acid bacteria in mixed fermentation, so that the bran is selected as the optimal carbon source.
As can be seen from FIG. 4, when the nitrogen source is corn steep liquor dry powder, the number of viable bacteria and the number of spores of Clostridium butyricum are highest, and respectively reach 5.6 multiplied by 107cfu/mL and 3.5X 107cfu/mL, and the viable count of Bacillus coagulans is much higher than other nitrogen sources. In view of the use of complex nitrogen sources in clostridium butyricum optimized media in many studies, corn steep liquor dry powder was complexed with other nitrogen sources in combination with the results of this experiment, as shown in table 2.
TABLE 2 Complex Nitrogen Source optimization results
Note: the proportion of the corn steep liquor dry powder to other nitrogen sources is 1:1, the total content of the nitrogen sources is 15g/L, and the contrast is 15 g/L.
As can be seen from Table 2, in addition to the combination of the dry corn steep liquor powder and the fish meal, the viable count of Clostridium butyricum when the dry corn steep liquor powder is compounded with other nitrogen sources is reduced to a different extent than that when the dry corn steep liquor powder is used alone, and only when the dry corn steep liquor powder is compounded with the fish meal, the viable count and the spore count of Clostridium butyricum are superior to those of a control group, and the viable count of Bacillus coagulans in the combination is higher than those of other combinations. Therefore, the corn steep liquor dry powder and the fish meal are selected to be compounded as an optimal nitrogen source.
The optimal types of the carbon source and the nitrogen source are determined through a single-factor experiment, and the concentration and the proportion of the optimal carbon source and the optimal nitrogen source also have great influence on the growth of the thalli, so an orthogonal experiment is designed to further research the concentration and the proportion of the carbon source and the nitrogen source, an L9(33) orthogonal table is designed by SPSS 20.0 software and shown as a table 3, and the results are analyzed and shown as a table 4.
TABLE 3 orthogonal test factor horizon
TABLE 4 results of orthogonal experiments
Note: a1, B1 and C1 correspond to the first level of each substance in the table, for example, A1 represents bran added at 10 g/L.
As can be seen from Table 4: the influence of the viable count of Clostridium butyricum is C>B>A shows that the dry corn steep liquor powder has a large influence on the number of viable bacteria of clostridium butyricum, and the influence of the number of spores of clostridium butyricum is A>C>B, the influence of the bran on the transsporidium of the clostridium butyricum is larger, and the influence of the viable count of the bacillus coagulans is B>A>C, the influence of the fish meal on the number of live bacillus coagulans is large. The optimized experimental conditions obtained by comparing the K values are as follows: A1B3C3, but the process condition is not in the experiment carried out, the result of the verification test is that the viable count of clostridium butyricum is 8.8 multiplied by 107cfu/mL, Clostridium butyricum spore number 7.8X 107cfu/mL, viable count of Bacillus coagulans 8.1 × 107cfu/mL。
The metal ions contained in the inorganic salt are the cofactors of many enzymes in the microorganism, and have an important effect on the growth of the microorganism. The results of the influence of several common inorganic salts on the growth of the bacteria are shown in FIG. 5, wherein the addition amounts of sodium chloride and potassium chloride are respectively 5g/L, the addition amounts of dipotassium hydrogen phosphate and calcium carbonate are respectively 1g/L, the addition amounts of sulfuric acid and manganese magnesium sulfate are respectively 0.3g/L, the addition amount of sodium acetate trihydrate is 3g/L, and the control is no inorganic salt.
As can be seen from FIG. 5, the addition of dipotassium hydrogen phosphate is the most effective, and has a promoting effect on the growth of Clostridium butyricum and promotes the transformation of spores, and the viable count of Bacillus coagulans is slightly higher than that of the control group. The optimization of the composite inorganic salt was performed by selecting three inorganic salts with better results, namely, dipotassium hydrogen phosphate, sodium acetate trihydrate, manganese sulfate and magnesium sulfate, and the results are shown in table 5.
TABLE 5 composite inorganic salt optimization results
Note: the combination is as follows: potassium dihydrogen phosphate and magnesium sulfate; combining two: monopotassium phosphate and manganese sulfate; combining three components: potassium dihydrogen phosphate and sodium acetate trihydrate, control is potassium hydrogen phosphate alone.
As can be seen from Table 5, the indexes of the combination of potassium dihydrogen phosphate and manganese sulfate are better than those of the control group, and the viable count of Clostridium butyricum reaches 1.1 × 107cfu/mL, the spore transformation rate reaches 92.2%, so potassium dihydrogen phosphate and manganese sulfate are selected as the best inorganic salts.
The experimental result of the previous step shows that the monopotassium phosphate and the manganese sulfate are the best inorganic salts, the SPSS 20.0 software is used for designing an L9(32) orthogonal table to explore the concentration and the proportion of the monopotassium phosphate and the manganese sulfate, the results are analyzed, the factors and the levels of the orthogonal test are shown in a table 6, and the results of the extremely poor and K value analysis are shown in a table 7.
TABLE 6 orthogonal test factor horizon
TABLE 7 results of orthogonal experiments
Note: a1 and B1 correspond to the first levels of the substances in the tables, and A1 represents that the addition amount of dipotassium hydrogen phosphate is 1 g/L.
By performing range analysis on the experiment, the following results can be obtained: the influence of the number and the number of the live bacteria of the clostridium butyricum and the number of the live bacteria of the bacillus coagulans is A>And B, showing that the dipotassium phosphate has great influence on the number and the number of spores of clostridium butyricum and the number of viable bacillus coagulans. The optimal experimental conditions obtained by comparing the K values are as follows: A1B3, i.e. the content of dipotassium hydrogen phosphate is 1.0g/L, the content of manganese sulfate is 0.5g/L, and the viable count of the obtained clostridium butyricum is 1.3 multiplied by 108cfu/mL, the spore rate reaches 92.3 percent.
From the above results, it can be seen that the optimal fermentation medium of the present invention comprises bran 10g/L, peptone 15g/L, corn steep liquor dry powder 15g/L, dipotassium hydrogen phosphate 1g/L, and manganese sulfate 0.5 g/L.
Example 3 optimization of culture conditions for Mixed fermentations
And optimizing the mixed fermentation culture condition on the basis of the optimal fermentation culture medium.
A 250mL Erlenmeyer flask was charged with 40% volume of medium, initial pH 7.0; and inoculating 8% clostridium butyricum and 4% bacillus coagulans seeds after sterilization and cooling, and detecting the concentration of the bacteria after static culture at 37 ℃ for 36 h.
(1) The fermentation temperatures were selected from 28 ℃, 33 ℃, 37 ℃, 40 ℃ and 45 ℃, and the effect of the fermentation temperatures on the fermentation was examined, and the results are shown in FIG. 6. As can be seen from FIG. 6, the temperature has a great influence on the growth of the strain in the mixed fermentation process, when the temperature is in the range of 28-37 ℃, the bacterial concentrations of Clostridium butyricum and Bacillus coagulans are gradually increased along with the temperature rise, and when the temperature is 37 ℃, the Clostridium butyricum has a large influence on the growth of the strainThe number of live bacteria of the bacillus coagulans and the bacillus coagulans reaches the highest and is respectively 1.38 multiplied by 108
cfu/mL and 8.5X 107cfu/mL, the number of viable bacteria of clostridium butyricum is obviously reduced after the temperature exceeds 40 ℃. The appropriate temperature for single fermentation of clostridium butyricum is 37 ℃, the appropriate temperature for bacillus coagulans is 37-50 ℃, and the mixed fermentation of clostridium butyricum and bacillus coagulans needs to combine the growth characteristics of two strains. The temperature optimization result of the experiment shows that the temperature of 37 ℃ is the optimum temperature for mixed fermentation.
(2) The initial pH was varied to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and the effect of pH on fermentation was examined, as can be seen from the results in fig. 7: the initial pH is within the range of 5.0-7.0, the viable count of clostridium butyricum gradually increases along with the increase of the initial pH, and the viable count of clostridium butyricum reaches the highest when the initial pH is 7.0. At an initial pH of 5.0, bacillus coagulans was able to grow, while clostridium butyricum almost stopped growing, indicating that in a mixed fermentation of the two, a lower pH was not conducive to the growth of clostridium butyricum. Therefore, the optimum initial pH for the mixed fermentation is 7.0.
(3) Selecting five gradients of 20%, 30%, 40%, 50% and 60% to examine the influence of the liquid filling coefficient on the fermentation. From the results in fig. 8, it can be seen that the viable count of clostridium butyricum and bacillus coagulans reaches the highest when the liquid filling coefficient is 40%, the viable count of clostridium butyricum is maintained at a higher level when the liquid filling coefficient is greater than 40%, while the viable count of bacillus coagulans is greatly reduced, and although bacillus coagulans is facultative anaerobe, a great deal of research shows that higher dissolved oxygen is beneficial to the growth of bacillus coagulans. The clostridium butyricum is a strict anaerobe, and the growth of the clostridium butyricum is facilitated due to a higher liquid filling coefficient. In the experiment, the liquid filling coefficient of 40% is selected as the optimal liquid filling coefficient of mixed fermentation, and under the liquid filling coefficient, higher thallus concentration can be obtained, and the cost is reduced.
(4) The inoculation ratio is changed, and the influence of different inoculation ratios on the fermentation is examined. The inoculum size of clostridium butyricum and bacillus coagulans was inoculated into the fermentation medium at 9 different ratios, and the results are shown in table 8.
TABLE 8 Effect of inoculation ratio of strains on growth of strains
Note: the inoculation ratio of 4:6 is that the inoculation amount of the bacillus coagulans is 4 percent, the inoculation amount of the clostridium butyricum is 6 percent, and the inoculation is carried out simultaneously.
As can be seen from Table 8, the cell concentration of Clostridium butyricum decreased as a whole with the increase of the inoculation amount of Bacillus coagulans, and the reason for this was that the growth of Clostridium butyricum was inhibited because the culture medium was depleted of nutrients by excessive Bacillus coagulans. When 4% and 8% of bacillus coagulans and clostridium butyricum are inoculated respectively, the viable count and the spore count of the clostridium butyricum are highest, and the viable count of the bacillus coagulans is higher.
(5) According to the fermentation process, the bacterial concentration is sampled and detected every 4 hours within 0-48 hours, and the pH value of the bacterial liquid is measured to determine the culture time, and the result is shown in FIG. 9. As can be seen from FIG. 9, in 0-12 h, the Bacillus coagulans rapidly propagates by using the nutrients in the culture medium and consumes the oxygen remaining in the culture medium to provide an anaerobic environment for the growth of Clostridium butyricum, and the Clostridium butyricum begins to propagate and rapidly enters a logarithmic phase at 12h, and the pH value reaches the lowest value at this time. And in 12-16 h, the clostridium butyricum and the bacillus coagulans grow simultaneously, the bacillus coagulans provides a strict anaerobic environment for the growth of the clostridium butyricum, and the clostridium butyricum provides growth factors such as vitamins for the bacillus coagulans. The clostridium butyricum begins to sporulate in 20h, and the spore transfer rate reaches the maximum value in 40h, but the absolute value of the spore number is slightly lower than 36 h. Taken together, 36h was taken as the end point of the mixed fermentation.
Example 4 feed Process optimization
(1) Influence of different feed supplement carbon-nitrogen ratios on fermentation
A10L fermentation tank is filled with a culture medium with an optimized volume of 40%, the initial pH value is adjusted to 7.0, sterilization is carried out for 30min at 121 ℃, after cooling, 8% clostridium butyricum and 4% bacillus coagulans are inoculated, standing culture is carried out at 37 ℃, the rotational speed is adjusted to 150rpm after a logarithmic phase, 6g/L glucose starts to be supplemented, continuous feeding is carried out for 8h, secondary feeding is carried out immediately after first feeding is finished, feeding liquid is 6g/L, peptone is 2g/L, and composite feeding liquid consisting of dipotassium hydrogen phosphate 0.5g/L is fed till the end of a logarithmic phase, and the contents and the proportion of a carbon source and a nitrogen source of the composite feeding liquid are researched. Sampling for 44h at regular time in the fermentation process, and detecting the thallus concentration and the residual sugar content of the fermentation liquor. The results are shown in FIG. 10. As can be seen from FIG. 10, when the feed solution only contains glucose, the number of viable bacteria of Clostridium butyricum is increased, and the increase of Bacillus coagulans is large. After the nitrogen sources with different proportions are added, the number of the live bacteria of the clostridium butyricum and the spore rate are both improved, when the carbon-nitrogen ratio is 2:1, the number of the live bacteria of the clostridium butyricum is the highest, the spore rate of the clostridium butyricum is also higher, and the total number of the live bacteria in the fermentation liquor is also the highest.
(2) Influence of pH and feed on fermentation during fermentation
In the fermentation process, the pH was controlled to 6.0 or not adjusted, and the fermentation was supplemented or not supplemented, and the influence of pH and supplementation on the fermentation was examined, with specific results shown in Table 9.
TABLE 9 fed-batch and pH adjusted fermentation results
Note: the control 1 is pH 6.0 controlled without feeding, and the control 2 is natural fermentation without feeding and pH adjustment.
As can be seen from Table 9, the feeding and pH control have an important influence on the fermentation results, and the spore rate of Clostridium butyricum can be effectively increased and the viable count of Bacillus coagulans can be increased by feeding and pH control.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, but rather as the subject matter of any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention.
Claims (1)
1. A liquid mixed fermentation method of Clostridium butyricum and Bacillus coagulans is characterized by comprising the following steps:
(1) preparing a fermentation medium, filling the fermentation medium into a fermentation tank, adjusting the initial pH of the fermentation medium to 6.0-7.5, and sterilizing at 121 ℃ for 30 min;
(2) carrying out liquid culture on clostridium butyricum and bacillus coagulans strains to obtain inoculated liquid; the inoculation liquid of the clostridium butyricum is obtained by carrying out deep static culture on a liquid at 37 ℃ for 12-16 h, and the inoculation liquid of the bacillus coagulans is obtained by carrying out stirring culture at 37 ℃ for 16-18 h;
(3) inoculating the inoculation liquid of the clostridium butyricum and the bacillus coagulans into a fermentation tank, and standing for culture at the fermentation temperature of 33-37 ℃ for 32-44 hours; starting a fermentation tank for stirring in the fermentation process, adjusting the rotation speed to 20-150 rpm, and performing first feeding, wherein the components of a feeding liquid are 2-10 g/L of glucose and last for 8 hours; after the first feeding, performing second feeding, wherein the components of a feeding liquid are a composite feeding liquid consisting of 1-6 g/L of glucose, 1-6 g/L of peptone and 0.1-1 g/L of dipotassium hydrogen phosphate, and feeding till the end of a logarithmic phase;
the fermentation medium comprises the following components: 0.5-20 g/L of bran, 0.5-25 g/L of peptone, 1-25 g/L of corn steep liquor dry powder, 0.1-5 g/L of dipotassium phosphate and 0.1-5 g/L of manganese sulfate;
the liquid filling coefficient of the fermentation tank is 20-60%;
the inoculation amount of the clostridium butyricum is 8 percent, and the inoculation amount of the bacillus coagulans is 4 percent.
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| CN112625980A (en) * | 2021-01-15 | 2021-04-09 | 驻马店华中正大有限公司 | Process for producing butyric acid by co-culture fermentation of bacillus amyloliquefaciens and clostridium butyricum |
| CN113265440A (en) * | 2021-05-25 | 2021-08-17 | 广东大泽农生物科技股份有限公司 | Compound acidifier and preparation method and application thereof |
| CN113604380A (en) * | 2021-07-15 | 2021-11-05 | 山东益昊生物科技有限公司 | Method for producing composite microbial inoculum by fermenting clostridium butyricum centrifugal waste liquid, corresponding composite microbial inoculum and application thereof |
| CN113652373B (en) * | 2021-08-31 | 2023-03-24 | 河南金百合生物科技股份有限公司 | Bacillus and application thereof |
| JP6994797B1 (en) | 2021-09-09 | 2022-01-14 | 有限会社ラヴィアンサンテ | How to grow butyrate-producing bacteria and how to manufacture food additives or feed additives |
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