CN116396874A - Combined fermentation method of clostridium butyricum - Google Patents
Combined fermentation method of clostridium butyricum Download PDFInfo
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- CN116396874A CN116396874A CN202310306684.7A CN202310306684A CN116396874A CN 116396874 A CN116396874 A CN 116396874A CN 202310306684 A CN202310306684 A CN 202310306684A CN 116396874 A CN116396874 A CN 116396874A
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- fermentation
- clostridium butyricum
- combined
- saccharomyces cerevisiae
- lactobacillus acidophilus
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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
Technical Field
The invention relates to a combined fermentation method of clostridium butyricum, belonging to the technical field of a microecological preparation production method.
Background
In recent years, the microecological preparation plays an important role in the fields of medical health care, food, feed, livestock and poultry cultivation and the like, and along with the gradual development of the limiting resistance, the microecological preparation is increasingly applied to the livestock and poultry cultivation field. Researches prove that the microecological preparation can maintain the balance of flora in animal intestinal tracts, prevent intestinal tract infection and improve the immunity of animal organisms. In addition, as probiotics can secrete a plurality of digestive enzymes, the microecological preparation plays a role in improving the utilization rate of feed, improving the production performance of animals and improving the quality of livestock and poultry products.
Clostridium butyricum has extremely strong intestinal function regulating effect, and is increasingly applied to feed additives due to the great advantages of the clostridium butyricum in the aspects of regulating the intestinal functions of animals, improving the growth performance of animals and enhancing the immunity. Lactobacillus acidophilus belongs to the genus Lactobacillus, and is clinically used for regulating intestinal flora balance and inhibiting proliferation of intestinal undesirable microorganisms. Saccharomyces cerevisiae can promote the absorption of nutrients in feed and improve the immunity of animal organisms, is also a single-cell protein with rich nutrition, and has the effects of shortening the feeding period, improving the meat quality and increasing the lean meat percentage.
Clostridium butyricum is a strict anaerobic bacterium, and in the fermentation process, the clostridium butyricum needs to be carried out in an anaerobic environment maintained by paraffin oil or inert gases such as nitrogen, carbon dioxide and the like, which increases the complexity of equipment and the difficulty of a method in the clostridium butyricum fermentation process and increases the production cost.
In order to solve the problems, chinese patent application publication No. CN111500508A discloses a liquid mixed fermentation method of clostridium butyricum and clostridium coagulans, which is used for preparing seed liquid of clostridium coagulans and clostridium butyricum and then inoculating the seed liquid into a liquid culture medium for fermentation culture. The mixed fermentation method can achieve viable count and spore count of clostridium butyricum of 5.8X10 8 cfu/mL and 5.3X10 8 cfu/mL。
The Chinese patent application with publication number of CN110241053A discloses a method for culturing clostridium butyricum by mixed fermentation, which comprises the steps of respectively culturing clostridium butyricum, bacillus subtilis, saccharomycetes and lactobacillus separately to obtain a single-strain culture solution, mixing according to a certain proportion, and carrying out mixed culture. The mixed fermentation culture method can achieve clostridium butyricum concentration of 1×10 9 cfu/mL。
However, the above methods of mixed fermentation are simultaneous inoculation, wherein the bacillus coagulans and the bacillus subtilis grow fast and consume too much nutrients, affecting the concentration of clostridium butyricum in the final fermentation product to some extent. Therefore, development of a fermentation method which is simple, low in cost and higher in clostridium butyricum concentration is a problem to be solved.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a combined fermentation method of clostridium butyricum, which avoids the problems that nitrogen or an anaerobic agent is required to provide anaerobic conditions in the anaerobic fermentation process of clostridium butyricum, the fermentation cost and equipment investment are increased, and the problem that the concentration of clostridium butyricum is lower in the mixed fermentation of clostridium butyricum in the prior art is solved.
In order to achieve the above purpose, the technical scheme of the combined fermentation method of clostridium butyricum in the invention is as follows:
a combined fermentation process for clostridium butyricum comprising the steps of: 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 beneficial effects of the technical scheme are that: the invention inoculates Saccharomyces cerevisiae, lactobacillus acidophilus and Clostridium butyricum into liquid culture medium at a time for joint fermentation culture. In the combined fermentation, the saccharomyces cerevisiae and lactobacillus acidophilus are sufficient to consume oxygen in a culture medium, an anaerobic environment is provided for the culture of clostridium butyricum, the consumed nutrient substances are less when the proliferation process is completed, more nutrients are distributed to the clostridium butyricum, and the viable count and the spore count of the clostridium butyricum in the mixed fermentation broth obtained after the fermentation is completed can reach 2.73X10 9 cfu/mL and 2.65X10 9 cfu/mL。
As a further improvement, the time-staggered inoculation is to inoculate Saccharomyces cerevisiae into a liquid culture medium firstly, inoculate lactobacillus acidophilus after 5-8 hours and inoculate clostridium butyricum after 12-16 hours. Preferably, the initial pH of the liquid culture medium is 6.8-7, the canning coefficient is 40-50%, the saccharomyces cerevisiae is inoculated into the liquid culture medium in an inoculum size of 4-5%, after culturing for 5-8 hours at 35-37 ℃, lactobacillus acidophilus is inoculated in an inoculum size of 2-3% for continuous culturing, after 12-16 hours, clostridium butyricum is finally inoculated in an inoculum size of 4-5%.
The beneficial effects of the technical scheme are that: the lactobacillus acidophilus is facultative anaerobic bacteria, can further consume oxygen in the system on the basis of saccharomycetes, and creates a strict anaerobic environment for the fermentation of clostridium butyricum.
As a further improvement, the viable count and spore count of clostridium butyricum in the final fermentation broth of the combined fermentation are not less than 2 multiplied by 10 9 cfu/mL。
The beneficial effects of the technical scheme are that: the invention uses the time-staggered inoculation of the saccharomyces cerevisiae, the lactobacillus acidophilus and the clostridium butyricum, can obviously improve the quantity of the clostridium butyricum, and the viable count and the spore count of the clostridium butyricum in the final combined fermentation liquor can reach 2.73X10 9 cfu/mL and 2.65X10 9 cfu/mL。
As a further improvement, the viable count of Lactobacillus acidophilus in the final fermentation broth of the combined fermentation is not less than 1×10 10 cfu/mL, the viable count of Saccharomyces cerevisiae is not less than 3×10 9 cfu/mL。
The beneficial effects of the technical scheme are that: the invention utilizes the time-staggered inoculation of the saccharomyces cerevisiae, the lactobacillus acidophilus and the clostridium butyricum, can maximally realize the reasonable utilization of resources, and the viable count of the saccharomyces cerevisiae and the lactobacillus acidophilus in the final combined fermentation liquor can reach 3.71 multiplied by 10 9 cfu/mL and 1.69×10 10 cfu/mL。
As a further improvement, the components of the liquid medium include: 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.
The beneficial effects of the technical scheme are that: the invention optimizes the components of the combined liquid culture medium based on the respective fermentation characteristics and production cost of Saccharomyces cerevisiae, lactobacillus acidophilus and Clostridium butyricum. The optimized formula can ensure the smooth progress of the mixed fermentation process, and the obtained viable count is higher and the cost is lower.
As a further improvement, the pH control range in the combined liquid fermentation process is 6.0-7.0.
The beneficial effects of the technical scheme are that: the pH is controlled between 6.0 and 7.0 in the process of combined liquid fermentation, so that the spore rate of clostridium butyricum can be effectively improved, and the viable count of clostridium butyricum, lactobacillus acidophilus and saccharomyces cerevisiae can be also improved.
As a further improvement, the fermentation canning liquid coefficient in the combined liquid state fermentation is 30-70%.
The beneficial effects of the technical scheme are that: while ensuring successful fermentation, the production cost is saved and the investment is reduced.
As a further improvement, the inoculation amount of clostridium butyricum, lactobacillus acidophilus and saccharomyces cerevisiae is 2-6%.
The beneficial effects of the technical scheme are that: the inoculation amount can ensure that the fermentation process is normally carried out, and the clostridium butyricum and the spore number in the final product can reach a higher level.
As a further improvement, the feeding is started 5-7 hours after the clostridium butyricum is inoculated, and the feeding is carried out until the logarithmic phase is finished.
The beneficial effects of the technical scheme are that: the feeding time and duration affect the final result of fermentation, and the spore rate of clostridium butyricum in mixed culture can be effectively improved according to the feeding scheme.
As a further improvement, the feeding comprises a first feeding and a second feeding, wherein the second feeding is directly started after the first feeding 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. Specifically, the solvent of the feed supplement liquid is water.
The beneficial effects of the technical scheme are that: the material is fed in batches according to the material feed liquid components of the material feed, so that the maximization of energy utilization can be effectively ensured.
As a further improvement, the combined fermentation method comprises inoculating fermentation centrifugate of combined fermentation into a solid state fermentation medium, and obtaining the feed additive through solid state fermentation.
The beneficial effects of the technical scheme are that: the fermentation centrifugal waste liquid of the combined fermentation contains rich nutrients such as protein, polysaccharide, inorganic salt and the like, and also contains thalli which are not thoroughly centrifuged, so that the waste of resources caused by wastewater treatment is reduced after the thalli are recycled, the pressure of environmental protection is reduced, the resources are utilized to the maximum extent, and the cost of related enterprises for the fermentation wastewater treatment is reduced.
As a further improvement, 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.
The beneficial effects of the technical scheme are that: the solid state fermentation culture medium contains a large amount of straws, so that the straws serving as byproducts are recycled, resources are saved, and meanwhile, the production cost is reduced for related enterprises.
As a further improvement, the conditions of the solid state fermentation are 30-37 ℃ for 3-7 d.
As a further improvement, the inoculation amount of the fermentation centrifugate is 2% -6% (v/m).
Drawings
FIG. 1 is a graph showing the growth curves of Clostridium butyricum, lactobacillus acidophilus and Saccharomyces cerevisiae in example 1 of the present invention;
FIG. 2 is the effect of carbon source on strain growth in example 2 of the present invention;
FIG. 3 is the effect of nitrogen source on strain growth in example 2 of the present invention;
FIG. 4 is the effect of inorganic salts on strain growth in example 2 of the present invention;
FIG. 5 is the effect of temperature on strain growth in example 3 of the present invention;
FIG. 6 is the effect of pH on strain growth in example 3 of the present invention;
FIG. 7 is the effect of loading factor on strain growth in example 3 of the present invention;
FIG. 8 is a graph showing the growth of each strain after the shake flask culture condition is optimized in example 3 of the present invention;
FIG. 9 is the effect of feed liquid carbon nitrogen ratio on strain growth during fermentation in a 10L tank in example 4 of the present invention;
in fig. 1-9: the left ordinate is for the concentration of clostridium butyricum and saccharomyces cerevisiae, and the right ordinate is for the concentration of lactobacillus acidophilus.
Detailed Description
The invention is further described in connection with the following detailed description, but the scope of the invention is not limited thereto; unless otherwise specified, all reagents, instruments, etc. used in the examples are commercially available.
The following examples and experimental examples are briefly described below for some of the biological materials, experimental reagents, experimental facilities, and the like:
culture medium:
the clostridium butyricum activation medium has the same components as the seed medium, and comprises the following components: 10g/L of peptone, 10g/L of beef extract, 3g/L of yeast powder, 5g/L of glucose, 1g/L of soluble starch, 5g/L of sodium chloride, 3g/L, L-cysteine hydrochloride, 0.5g/L of sodium acetate, 0.5g/L of agar and pH value of 6.8;
clostridium butyricum counting medium: 2.0% of agar powder is added into the RCM liquid culture medium;
the lactobacillus acidophilus activation culture medium and the seed culture medium have the same components and are all MRS liquid culture medium, and the lactobacillus acidophilus activation culture medium comprises the following components: 10g/L of casein peptone, 10g/L of beef extract, 5g/L of yeast extract, 20g/L of glucose, 5g/L of sodium acetate, 2g/L of citric acid diamine, 1g/L of tween 80, 2g/L of dipotassium hydrogen phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate heptahydrate, 20g/L of calcium carbonate and pH value of 6.8;
lactobacillus acidophilus counting medium: 2.0% of agar powder is added into MRS liquid culture medium;
the saccharomyces cerevisiae activation culture medium and the seed culture medium have the same components and are all YPD culture mediums, and the saccharomyces cerevisiae activation culture medium comprises the following components: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, pH 5.6;
saccharomyces cerevisiae counting medium: 2.0% agar powder was added to YPD liquid medium.
1. Specific example of combined fermentation method of clostridium butyricum
Example 1 determination of Strain growth Curve
The culturing mode of clostridium butyricum: liquid deep stationary culture, cone bottled liquid coefficient 60%, inoculum size 4%, sealing with 8 layers of gauze and 2 layers of kraft paper, stationary culturing at 37deg.C for 48 hr.
The culture mode of lactobacillus acidophilus: liquid deep stationary culture, cone bottle liquid coefficient 40%, inoculum size 4%, sealing with 8 layers of gauze, stationary culturing at 37deg.C for 48 hr.
The culture mode of the saccharomyces cerevisiae comprises the following steps: liquid shake flask culture, cone bottle liquid coefficient 40%, inoculum size 5%, and culture at 30deg.C 180r/min for 48 hr.
The strain activation and culture were performed according to the above method, and the growth curves were respectively drawn by sampling and measuring the number of viable bacteria at 4h intervals during the culture, as shown in FIG. 1.
As can be seen from FIG. 1, the three strain growth curves have high concentration of late logarithmic phase bacteria and vigorous vitality of bacteria, so that the culture times of clostridium butyricum, lactobacillus acidophilus and Saccharomyces cerevisiae seeds are respectively selected from 16h, 18h and 36h.
Example 2 optimization of the composition of the Co-liquid Medium in Mixed fermentation
The initial fermentation mode of mixed culture: according to the growth curves of clostridium butyricum, lactobacillus acidophilus and saccharomyces cerevisiae, taking an RCM culture medium as an initial combined culture medium, firstly inoculating the saccharomyces cerevisiae into the combined liquid culture medium with an inoculum size of 4%, inoculating the lactobacillus acidophilus into the combined liquid culture medium with an inoculum size of 4% after 6 hours, inoculating the clostridium butyricum into the combined culture medium with an inoculum size of 4% after 14 hours, carrying out stationary culture at a temperature of 37 ℃ with a cone-shaped bottled liquid coefficient of 50%, and sealing an cone-shaped bottle by 8 layers of gauze and 2 layers of kraft paper.
The time interval selection principle of the time-staggered inoculation is as follows: according to the growth curves of clostridium butyricum, lactobacillus acidophilus and saccharomyces cerevisiae shown in fig. 1, the time when the three strains enter the logarithmic phase is known, if the saccharomyces cerevisiae enters the logarithmic phase, the growth speed is fast and oxygen is consumed, at the moment, the best inoculation time for inoculating lactobacillus acidophilus with low oxygen demand is obtained, for example, more nutrient components are consumed after inoculation, and the nutrition left for the target clostridium butyricum is reduced.
Optimizing carbon sources glucose on the basis of an initial culture medium, and selecting 8 carbon sources of glucose, sucrose, lactose, maltose, soluble starch, corn starch, bran and soybean meal to explore the influence of different carbon sources on the growth of the combined culture strains.
The nitrogen source is preferably selected from peptone, tryptone, yeast extract, corn steep liquor dry powder, soybean meal powder, fish meal, beef extract, and yeast extract powder by using the selected optimized carbon source.
And (3) designing an orthogonal experiment according to the optimization results of the carbon source and the nitrogen source types, optimizing the concentrations of the carbon source and the nitrogen source, wherein the carbon source concentration is set to be three concentrations of 15g/L, 20g/L and 25g/L, and the nitrogen source concentration is set to be three concentrations of 10g/L, 20g/L and 30 g/L.
The inorganic salt is selected from magnesium sulfate, manganese sulfate, dipotassium hydrogen phosphate, sodium acetate trihydrate, calcium carbonate, sodium chloride and potassium chloride in initial test. On the basis of optimizing the inorganic salt types, orthogonal experiments are designed to optimize the inorganic salt concentration. In the optimization process, the number of live clostridium butyricum and spores thereof, the number of live lactobacillus acidophilus and the number of live saccharomyces cerevisiae are selected as investigation indexes, clostridium butyricum is taken as a main investigation object, and each group of experiments is repeated for 3 times.
2.1 optimization of carbon Source
As can be seen from fig. 2, when glucose is used as a carbon source, the number of live clostridium butyricum is much higher than that of other carbon sources; the most viable bacteria of lactobacillus acidophilus and saccharomyces cerevisiae when the carbon source is maltose; the number of live clostridium butyricum, lactobacillus acidophilus and saccharomyces cerevisiae is ideal when the bran is a carbon source. To further optimize the carbon source, glucose, maltose, sucrose, lactose were separately complexed with bran to explore the effect of the complexed carbon source on the growth of the species, and the results are shown in table 1.
TABLE 1 optimization results of composite carbon sources
Note that: the combination of glucose 12g/L and bran 8g/L; the second combination is 10g/L of maltose and 10g/L of bran; the third combination is 12g/L of sucrose and 8g/L of bran; the fourth combination is lactose 10g/L and bran 10g/L; the control was 20g/L glucose.
As can be seen from Table 1, the effect of the composite carbon source is more favorable for the three-bacteria combined culture than that of the single carbon source. When the composite carbon source is glucose and bran, the number of viable bacteria and spores of clostridium butyricum reach the maximum, and when the composite carbon source is maltose and bran, the number of viable bacteria of lactobacillus acidophilus and saccharomyces cerevisiae reach the maximum, and the number of viable bacteria and spores of clostridium butyricum are slightly lower than the number of viable bacteria and spores of clostridium butyricum. In comprehensive consideration, glucose and bran are selected as carbon sources for the combined culture.
2.2 optimization of Nitrogen Source
As can be seen from FIG. 3, when the nitrogen source is yeast extract, the viable count and the spore count of Clostridium butyricum are both highest, reaching 2.36×10 respectively 7 cfu/mL and 2.12X10 7 cfu/mL, and the viable count of Lactobacillus acidophilus is also much higher than other nitrogen sources. The nitrogen source isWhen peptone is used, the number of viable bacteria of Saccharomyces cerevisiae reaches the maximum value. According to the related literature, the compound nitrogen source can achieve better effect, so that the experimental result is shown in table 2 after the yeast extract is compounded with other nitrogen sources.
TABLE 2 optimization results of Complex Nitrogen Source
Note that: the ratio of the yeast extract to other nitrogen sources is 1:1, the nitrogen source content is 20g/L, and the contrast is 20g/L.
As is clear from Table 2, in the combination of yeast extract and peptone as the composite nitrogen source, the number of viable bacteria of Clostridium butyricum and the number of spores and the number of viable bacteria of Lactobacillus acidophilus all reached the maximum value; when the composite nitrogen source is yeast extract and fish meal, the number of viable bacteria of Saccharomyces cerevisiae reaches the maximum value. In the combination of the yeast extract and other nitrogen sources as the composite nitrogen source, the viable count of the three bacteria is improved to different degrees compared with that of the yeast extract when the yeast extract is singly used. Considering comprehensively, a composite nitrogen source of yeast extract and peptone is selected.
2.3 optimization of concentration ratio of carbon source and Nitrogen source
The types of the optimal carbon source and the nitrogen source are determined by a single factor experiment, the concentration and the proportion of the optimal carbon source and the nitrogen source have great influence on the growth of thalli, so that an orthogonal experiment is designed to further determine the optimal adding proportion of the composite carbon source and the nitrogen source, and SPSS 20.0 software is used for designing L9 (3 4 ) The orthogonality table is shown in table 3.
TABLE 3 orthogonal test factor level Table of concentration ratio of carbon source and Nitrogen source
The results were analyzed for extreme differences and K values and are shown in table 4.
TABLE 4 results of orthogonal experiments on concentration ratios of carbon sources and nitrogen sources
Note that: a is that 1 、B 1 、C 1 Respectively correspond to a first level of each substance in the table, e.g. A 1 The amount of glucose added was 15g/L.
As can be seen from table 4: the influence of the clostridium butyricum viable count is D & gtB & gtC & gtA, which indicates that the influence of peptone on the clostridium butyricum viable count is larger; the influence of the spore number of clostridium butyricum is B & gtD & gtA & gtC, which shows that the influence of the bran on clostridium butyricum spore transformation is larger; the influence of the live bacteria number of the lactobacillus acidophilus is B & gtC & gtA & gtD, which shows that the wheat bran has larger influence on the live bacteria number of the lactobacillus acidophilus; the influence of the number of the active bacteria of the saccharomyces cerevisiae is C & gtB & gtD & gtA, which shows that the influence of the yeast extract on the number of the active bacteria of the saccharomyces cerevisiae is larger. The optimal experimental conditions obtained by comparing the K values are as follows: a is that 3 B 2 C 3 D 3 However, the conditions of the method are not in the experiment, and the test result is verified that the viable count of clostridium butyricum is 4.67 multiplied by 10 8 cfu/mL, clostridium butyricum spore count 3.63×10 8 cfu/mL, lactobacillus acidophilus viable count 9.35×10 9 cfu/mL, the viable count of Saccharomyces cerevisiae is 2.27X10 8 cfu/mL。
2.4 optimization of inorganic salts
Although the effect of the inorganic salt on the fermentation thalli is not as good as that of the carbon source and the nitrogen source, the proper amount of the inorganic salt can promote the healthy and rapid growth and propagation of the thalli and increase the metabolite quantity of the thalli. As shown in FIG. 4, the effect of several common inorganic salts on the growth of the bacterial cells is that the addition amounts of sodium chloride and potassium chloride are 5g/L, the addition amounts of dipotassium hydrogen phosphate and calcium carbonate are 1g/L and 5g/L, the addition amounts of manganese sulfate and magnesium sulfate are 0.3g/L, and the addition amount of sodium acetate trihydrate is 3g/L, compared with the case that no inorganic salt is added.
As can be seen from FIG. 4, dipotassium hydrogen phosphate can promote clostridium butyricum growth and transformation, and the number of viable bacteria of lactobacillus acidophilus and saccharomyces cerevisiae reaches the maximum value when the inorganic salt is calcium carbonate. To further determine the composition of the inorganic salt, four inorganic salts with better results, namely dipotassium hydrogen phosphate, sodium acetate trihydrate, manganese sulfate, magnesium sulfate and calcium carbonate, were selected for optimizing the composite inorganic salt, and the results are shown in Table 5.
TABLE 5 optimization results of composite inorganic salts
Note that: combining dipotassium hydrogen phosphate and sodium acetate trihydrate; the second combination is dipotassium hydrogen phosphate and manganese sulfate; the combination III is dipotassium hydrogen phosphate and magnesium sulfate; combining dipotassium hydrogen tetraphosphate and calcium carbonate; the control was dipotassium hydrogen phosphate alone.
As is clear from Table 5, the viable count of Clostridium butyricum in combination III and combination IV was comparable, and the spore count of Clostridium butyricum in combination of dipotassium hydrogen phosphate and calcium carbonate was better, at which time the viable count of Clostridium butyricum reached 4.05X10 8 cfu/mL, the spore count reaches 3.75X10 8 . From the results, it was found that dipotassium hydrogen phosphate, magnesium sulfate and calcium carbonate can be selected as the optimal inorganic salt components in the co-culture medium.
The experimental results show that dipotassium hydrogen phosphate, magnesium sulfate and calcium carbonate are the best inorganic salts, and SPSS 20.0 software is used for designing L9 (3 3 ) The concentrations and ratios of the three are studied in the orthogonal table, and the results are analyzed, and the orthogonal test factors and levels are shown in table 6.
TABLE 6 orthogonal test factor level table for compound inorganic salt ratio
The results were analyzed for extreme differences and K values and are shown in table 7.
TABLE 7 orthogonal test results of the compound inorganic salt ratio
Note that: a is that 1 、B 1 、C 1 Respectively correspond to a first level of each substance in the table, e.g. A 1 Represents the addition amount of dipotassium hydrogen phosphate of 2g/L.
As can be seen from the experimental analysis: the influence of the live clostridium butyricum is C > B > A; the influence of the spore number is C > B > A; the influence of the viable count of the lactobacillus acidophilus is C & gtA & gtB; the influence of the number of the active bacteria of the saccharomyces cerevisiae is A > B > C. The best experimental condition obtained by comparing the K value is A 2 B 3 C 3 Namely, the addition amount of dipotassium hydrogen phosphate is 3.0g/L, the addition amount of magnesium sulfate is 0.8g/L, the addition amount of calcium carbonate is 8g/L, and the obtained clostridium butyricum viable count is 5.24X10 8 cfu/mL, spore count of 4.32X10 8 cfu/mL, the viable count of lactobacillus acidophilus is 1.05X10 10 cfu/mL, the number of active bacteria of Saccharomyces cerevisiae is 2.7X10 8 cfu/mL。
As shown in the results of optimizing the components of the combined liquid culture medium in the mixed fermentation, the optimal combined liquid culture medium comprises 25g/L of glucose, 6g/L of bran, 15g/L of peptone, 15g/L of yeast extract, 3g/L of dipotassium hydrogen phosphate, 0.8g/L of magnesium sulfate and 8g/L of calcium carbonate.
Example 3 optimization of Co-fermentation culture conditions
The combined fermentation culture conditions were optimized on the basis of the optimal fermentation medium obtained in example 2. A 250mL Erlenmeyer flask was filled with 50% volume of medium and the initial pH was 6.5; sterilizing, cooling, inoculating 4% clostridium butyricum, 4% lactobacillus acidophilus and 4% Saccharomyces cerevisiae seeds, standing at 37deg.C for 42 hr, and detecting thallus concentration.
3.1 optimization of fermentation temperature
The fermentation temperature is selected to be 30 ℃, 33 ℃,37 ℃,40 ℃,42 ℃ and 45 ℃ for investigationThe effect of temperature on fermentation is shown in FIG. 5. As can be seen from FIG. 5, the temperature has a remarkable effect on the growth of strains during the combined fermentation, and when the temperature is within the range of 30-37 ℃, the concentration of the clostridium butyricum, lactobacillus acidophilus and Saccharomyces cerevisiae gradually increases with the increase of the temperature, and when the temperature is 33 ℃, the number of viable bacteria of Saccharomyces cerevisiae reaches the maximum value of 2.45×10 8 cfu/mL. The number of live clostridium butyricum and lactobacillus acidophilus reaches the maximum value at 37 ℃, and the number is 4.96 multiplied by 10 respectively 8 cfu/mL and 9.14X10 9 cfu/mL. The viable count of clostridium butyricum is obviously reduced after the temperature exceeds 42 ℃. In combination with related documents and experimental study results, the growth characteristics of the three bacteria are comprehensively considered during the combined fermentation, so the temperature of the combined fermentation of the three bacteria is set to be 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 strain growth during the combined fermentation was examined, as can be seen from the results of FIG. 6: at an initial pH of 5.5, clostridium butyricum hardly grows, whereas in the range of 5.5 to 6.8, the number of viable clostridium butyricum gradually increases with the increase of the initial pH, and at an initial pH of 6.8, the number of viable clostridium butyricum reaches the highest. Lactobacillus acidophilus can grow at an initial pH of 5.5, and the number of viable bacteria reaches a maximum at an initial pH of 5.7. The growth curve of the saccharomyces cerevisiae along with the change of the pH value is more gentle, and when the initial pH value is 6.5, the number of the active bacteria of the saccharomyces cerevisiae reaches the maximum value. Referring to the relevant literature and combining the research results of the experiment, the optimal initial pH of the three-bacterium combined fermentation is set to be 6.8.
3.3 optimization of liquid filling coefficient
Five gradients of 30%, 40%, 50%, 60% and 70% are selected to examine the influence of the liquid loading coefficient on the combined fermentation. As can be seen from FIG. 7, when the loading factor was 50%, the viable count of Clostridium butyricum and Lactobacillus acidophilus reached the highest, and when the loading factor was 40%, the viable count of Saccharomyces cerevisiae reached the highest, but the viable count of Clostridium butyricum was slightly decreased, and the optimal loading factor of the three bacteria combined fermentation was set to 50% in view of the fact that too high loading factor was unfavorable for the growth of Clostridium butyricum.
3.4 optimization of inoculum size
The inoculum size of Clostridium butyricum, lactobacillus acidophilus and Saccharomyces cerevisiae was used as three factors, each of which was set to 3 levels, as per L9 (3 3 ) The orthogonality table is tested as in table 8.
TABLE 8 level of inoculum size orthogonal test factors
The results of the orthogonal experiments were analyzed by SPSS 20.0 software and the best inoculation ratio optimization results are shown in Table 9.
TABLE 9 results of inoculum size orthogonality test
Note that: a is that 1 、B 1 、C 1 Respectively correspond to a first level of each substance in the table, e.g. A 1 Represents 2% of the inoculation amount of clostridium butyricum.
As shown by the extremely poor analysis, the influence on the viable count of clostridium butyricum is C > A > B; the influence on the spore number of clostridium butyricum is A > C > B; the influence on the viable count of lactobacillus acidophilus is A > B > C; the influence on the number of active bacteria of the saccharomyces cerevisiae is A > C > B. The best experimental condition obtained by comparing the K value is A 3 B 1 C 2 That is, the inoculation amount of clostridium butyricum was 5%, the inoculation amount of lactobacillus acidophilus was 2%, and the inoculation amount of saccharomyces cerevisiae was 4%. As a result of the test conducted by this inoculation, the viable count of Clostridium butyricum was 5.12X10 8 cfu/mL, spore count of 4.23×10 8 cfu/mL, viable count of Lactobacillus acidophilus is 1.01X10 10 cfu/mL, the viable count of Saccharomyces cerevisiae is 2.41×10 8 cfu/mL。
3.5 optimization of fermentation time
According to the fermentation method, saccharomyces cerevisiae is inoculated into a combined liquid culture medium for 6 hours with an inoculum size of 4%, lactobacillus acidophilus is inoculated into the combined liquid culture medium with an inoculum size of 2%, and clostridium butyricum is inoculated into the combined culture medium with an inoculum size of 5% after 14 hours. The cell concentration was sampled and measured every 4 hours over 0 to 48 hours to determine the incubation time, and the results are shown in FIG. 8. As can be seen from FIG. 8, during 4-20 hours, saccharomyces cerevisiae rapidly breeds by utilizing the nutrient components in the culture medium, and during 8-20 hours, lactobacillus acidophilus rapidly grows, and the two bacteria consume the residual oxygen in the culture medium to provide an anaerobic environment for the growth of Clostridium butyricum while propagating in large quantities. And the clostridium butyricum starts to reproduce in 18 hours and rapidly enters a logarithmic phase, and grows together with lactobacillus acidophilus and saccharomyces cerevisiae in 20-30 hours, wherein the lactobacillus acidophilus and the saccharomyces cerevisiae provide a nutritional environment for the growth of the clostridium butyricum, and the clostridium butyricum provides growth factors such as vitamins for the other two bacteria. Clostridium butyricum starts sporulation at 34h and the number of viable bacteria reaches a maximum at 42h, but the absolute value of the spore rate is slightly lower than 40h at this time. Considering together, 42h was taken as the end point of the mixed fermentation.
Example 4 optimization of the feed method
The 10L fermentation tank is filled with 50% volume-optimized combined liquid culture medium, the initial pH is adjusted to 6.8, sterilization is carried out at 121 ℃ for 30min, dissolved oxygen is set to 100% after cooling, 4% of saccharomyces cerevisiae seeds are inoculated, when the dissolved oxygen is reduced to 60% after culturing at 33 ℃ and 160rpm for 6h, 2% of lactobacillus acidophilus is inoculated, and after standing culturing at 37 ℃, the dissolved oxygen is reduced to 10% after 8h, 5% of clostridium butyricum is inoculated, and standing culturing is carried out at 37 ℃. During the stationary culture, stirring was intermittently performed at 50rpm for 2min every 20min to prevent culture precipitation. And (3) 10g/L glucose is fed in 20h, continuous feeding is carried out for 8h, secondary feeding is carried out immediately after the primary feeding, the feeding liquid is a composite feeding liquid consisting of 8g/L glucose, 2g/L peptone and 1g/L dipotassium hydrogen phosphate, and the carbon source and nitrogen source contents and proportions of the composite feeding liquid are explored until the logarithmic phase is finished. Samples were taken at regular intervals to 48h during the fermentation, and the cell concentration and residual sugar content of the fermentation broth were measured, and the results are shown in FIG. 9. As can be seen from fig. 9, the carbon source in the feed solution has a large influence on saccharomyces cerevisiae, and the nitrogen source has a large influence on lactobacillus acidophilus. When the feed liquid has glucose only, the number of the clostridium butyricum viable bacteria is increased, after nitrogen sources with different proportions are added, the number of the clostridium butyricum viable bacteria and the spore rate are improved, when the carbon nitrogen ratio is 2:1, the number of the clostridium butyricum viable bacteria reaches the highest, the spore rate of the clostridium butyricum is also higher, and at the moment, the number of the viable bacteria in the fermentation liquid is also the highest. Comprehensively considering that the carbon-nitrogen ratio of the feed supplement liquid in the combined fermentation of clostridium butyricum, lactobacillus acidophilus and saccharomyces cerevisiae is 2:1.
In the fermentation process, the pH is controlled to be 6.8 or the pH is not adjusted, the material is fed or not fed in the fermentation process, the influence of the pH and the material fed in the fermentation process on the fermentation is examined, and the specific results are shown in Table 10.
TABLE 10 fed-batch and pH adjusted fermentation results
Note that: control 1 was pH 6.8 adjusted and controlled without feed supplementation, control 2 was natural fermentation without feed supplementation and pH adjustment.
As can be seen from Table 10, the feed and pH control have an important effect on the fermentation results, and the feed and pH control can effectively increase the spore yield of Clostridium butyricum and the viable count of Clostridium butyricum, lactobacillus acidophilus and Saccharomyces cerevisiae.
Example 5 recovery and utilization of Combined fermentation centrifugal waste liquid
Centrifuging the combined fermentation broth at 4000rpm for 10min, and spray drying to obtain bacterial powder containing Clostridium butyricum 1.25X10 in supernatant 7 cfu/mL, lactobacillus acidophilus 2.76X10 8 cfu/mL, saccharomyces cerevisiae 2.54×10 6 cfu/mL, can be used as seed liquid for solid feed fermentation.
Preparing a solid medium: firstly, crushing peanut, corn and wheat straw to 50 meshes. Then, the contents designed in the example are uniformly mixed with peanut straw, corn straw, wheat straw, bran, bean pulp, magnesium sulfate, calcium carbonate and dipotassium hydrogen phosphate, and sterilized at 121 ℃ for 20min.
Taking 5.0Kg of sterilized solid fermentation medium, filling into fermentation bags with one-way air release valves, adding 500g of supernatant into the bags according to different proportions, stirring uniformly, sealing, placing into an incubator, culturing at 37 ℃ for 7 days, and mixing the materials uniformly by shaking every day.
The bran bean pulp ratio, the feed water ratio and the strain inoculation amount in the solid state fermentation medium are important influencing factors in the solid state feed fermentation process, and the following process optimization research is carried out according to the literature and experience accumulated in the subject.
5.1, wheat bran bean pulp ratio, feed water ratio and supernatant inoculation amount optimization
In order to achieve the best solid state fermentation result, three factors of wheat bran bean pulp ratio, feed water ratio and supernatant inoculation amount are selected according to L9 (3 3 ) Orthogonal table an orthogonal test was performed as in table 11.
TABLE 11 orthogonal test factor level Table of wheat bran soybean meal ratio, feed water ratio and supernatant inoculum size
The results of the orthogonal experiments were analyzed using SPSS 20.0 software and the best optimization results are shown in table 12.
Table 12 results of orthogonal experiments on wheat bran soybean meal ratio, feed water ratio and supernatant inoculum size
Note that: a is that 1 、B 1 、C 1 Respectively correspond to a first level of each substance in the table, e.g. A 1 The ratio of the representative bran bean pulp is 1:2.
As can be seen from the extremely poor analysis, the influence of each factor on the number of live bacteria of clostridium butyricum is A > C > B, the influence of each factor on the number of live bacteria of lactobacillus acidophilus is C > A > B, and the influence of each factor on the number of live bacteria of saccharomyces cerevisiae is C > B > A. The best experimental condition obtained by comparing the K values is A 2 B 1 C 2 The ratio of bran to bean pulp is 1:3, the feed water ratio is 1:0.5, the inoculation amount of supernatant fluid is 15%, and the verification test is carried out under the condition, so that the result is that: the viable count of clostridium butyricum is 3.93 multiplied by 10 7 cfu/g, live bacteria number of Lactobacillus acidophilus is 7.51X10 8 cfu/g, the viable count of Saccharomyces cerevisiae is 7.94X10 6 cfu/g。
5.2, optimizing the adding proportion of wheat, corn and peanut straws
After the fermentation conditions are obtained, three factors of the wheat straw adding proportion, the corn straw adding proportion and the peanut straw adding proportion are selected based on the conditions, and the fermentation conditions are calculated according to L9 (3 3 ) Orthogonal table an orthogonal test was performed as in table 13.
TABLE 13 orthogonal test factor level Table for wheat, corn and peanut straw addition ratios
The results of the orthogonal experiments were analyzed using SPSS 20.0 software and the best optimization results are shown in table 14.
Table 14 results of orthogonal test of the addition ratio of wheat, corn and peanut straw
Note that: a is that 1 、B 1 、C 1 Respectively correspond to a first level of each substance in the table, e.g. A 1 The addition ratio of the representative wheat straw is 15%.
As can be seen from the extremely poor analysis, the influence of each factor on the number of live bacteria of clostridium butyricum is B & gtC & gtA, the influence of each factor on the number of live bacteria of lactobacillus acidophilus is C & gtB & gtA, and the influence of each factor on the number of live bacteria of saccharomyces cerevisiae is A & gtB & gtC. The optimal adding proportion of the wheat straw, the corn straw and the peanut straw can be obtained by comparing the K values is A 1 B 2 C 3 Namely, the adding proportion of the wheat straw is 5%, the adding proportion of the corn straw and the peanut straw is 7%, and the verification test is carried out under the condition, so that the result is that: the viable count of clostridium butyricum is 2.63 multiplied by 10 8 cfu/g, viable count of Lactobacillus acidophilus is 4.62X10 9 cfu/g, the viable count of Saccharomyces cerevisiae is 2.56X10 8 cfu/g。
5.3 optimization of the addition amount of inorganic salts
In order to further optimize the addition amount of the inorganic salt, three factors of the addition amounts of magnesium sulfate, dipotassium hydrogen phosphate and calcium carbonate are selected based on the above optimization conditions, according to L9 (3 3 ) Orthogonal table an orthogonal test was performed as in table 15.
TABLE 15 level of inorganic salt addition orthogonal test factors
The results of the orthogonal experiments were analyzed using SPSS 20.0 software and the best optimization results are shown in table 16.
TABLE 16 results of orthogonal test on the addition amount of inorganic salts
Note that: a is that 1 、B 1 、C 1 Respectively correspond to a first level of each substance in the table, e.g. A 1 Represents the addition amount of magnesium sulfate to be 0.1g/L.
As can be seen from the extremely poor analysis, the influence of each factor on the number of live bacteria of clostridium butyricum is A > B > C, the influence of each factor on the number of live bacteria of lactobacillus acidophilus is C > B > A, the influence of each factor on the number of live bacteria of saccharomyces cerevisiae is B > A > C, and the optimal addition ratio of magnesium sulfate, dipotassium hydrogen phosphate and calcium carbonate is A by comparing the K values 1 B 2 C 3 Namely, 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, and the results of the verification test under this condition were: the viable count of clostridium butyricum is 4.86 multiplied by 10 8 cfu/g, viable count of Lactobacillus acidophilus is 6.65X10 9 cfu/g, the viable count of Saccharomyces cerevisiae is 4.57×10 8 cfu/g。
In conclusion, the best experimental conditions for solid state fermentation by utilizing the centrifugal supernatant and the crop byproducts are obtained by the orthogonal experiments, namely, the wheat bran and bean pulp ratio is 1:3 (wheat bran 20%, bean pulp 60%), the adding proportion of the wheat straw is 5%, the adding proportion of the corn straw and the peanut straw is 7%, the adding amount of the magnesium sulfate is 0.1%, the adding amount of the dipotassium hydrogen phosphate is 0.3%, and the adding amount of the calcium carbonate is 1%. The feed water ratio was 1:0.5, and the inoculum size of the supernatant was 15%. After solid state fermentation, the viable count of clostridium butyricum in the solid state fermentation product reaches 4.86 multiplied by 10 8 cfu/g, the viable count of lactobacillus acidophilus reaches 6.65X10 9 cfu/g, the viable count of the saccharomyces cerevisiae reaches 4.57 multiplied by 10 8 cfu/g。
2. Experimental example
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 the application, and the process optimization was performed on the combined liquid fermentation method, and the obtained results are shown in table 17.
Table 17 comparison of fermentation results of different fermentation strains in Combined liquid fermentation
As is clear from Table 17, the addition of Lactobacillus acidophilus further consumed oxygen during fermentation, provided a good environment for fermentation of Clostridium butyricum, and increased the viable count and spore count of Clostridium butyricum in the final fermentation product.
Experimental example 2 comparison of solid state fermentation effects
The culture medium formulation and fermentation process optimized in examples 2-4 were subjected to liquid fermentation, and the resulting broth was centrifuged to obtain a supernatant for use in solid feed fermented seeds.
The supernatants were inoculated in 15% portions into the solid fermentation medium optimized in example 5, respectively, and simultaneously inoculated in 15% portions into a conventional solid medium (comprising 24.7% bran, 74.1% soybean meal, 0.1% magnesium sulfate, 0.3% dipotassium hydrogen phosphate, and 1% calcium carbonate) and sealed with a sealing film, and placed in an incubator at 37℃for culturing 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 conventional solid state medium, the viable count of Clostridium butyricum, lactobacillus acidophilus and Saccharomyces cerevisiae obtained by solid state fermentation using peanut straw, corn straw and wheat straw as a part of the medium components was comparable to the conventional solid state fermentation results. From this, it is found that a preferable fermentation result can be achieved by using straw instead of a part of the medium components, but at a lower cost.
Experimental example 3 animal feeding experiments of solid state fermentation products
The experimental example is used for explaining the influence of the solid fermentation product on the growth state and the immunity of calves.
30 healthy calves with the age of 50 days (80 Kg) are selected and randomly divided into 3 groups, 10 calves are fed to a control group, 0.5g/Kg of solid fermentation product in the fifth embodiment is added daily according to the weight of the calves on the basis of feeding basic feed in an experimental group I, 1g/Kg of the complex microbial inoculum (prepared according to the preparation method described in the patent) in patent CN108048355A, clostridium butyricum fermentation product and clostridium butyricum solid fermentation method is added daily according to the weight of the calves on the basis of feeding basic feed in an experimental group II, the main components of the complex microbial inoculum comprise soybean meal, wheat bran, corn flour, alkaline protease, alpha-amylase and the like, the complex microbial inoculum is freely fed by adopting a funnel type feed trough, water supply is sufficient, and daily management is carried out according to the conventional feeding experiment for 35 days.
The basic feed comprises the following components: 44.5% of corn, 20% of soybean meal, 10% of cottonseed meal, 20% of bran, 1% of stone powder, 1.5% of calcium bicarbonate, 0.8% of salt, 1.2% of baking soda and 1% of premix.
Feed weights were weighed at the beginning of the experiment (day 0), day 7, day 21 and at the end of the experiment (day 35) and calf weights were weighed at the end of the experiment (day 35). The number of diarrhea calves was counted 9 am daily, and the average daily gain (average daily gain, ADG), average daily feed intake (average daily feed intake, ADFI) and feed-to-weight ratio (feed: gain, F/G) were calculated.
Average Daily Gain (ADG) = (end-initial test weight)/days of test;
feed weight ratio = average daily feed intake/average daily gain;
diarrhea rate = number of diarrhea calves/(number of test calves x total days) x 100%,
wherein, experimental period diarrhea calf first = day 1 diarrhea calf number + day 2 diarrhea calf number + … … day 35 diarrhea calf number.
Statistical analysis and analysis of variance were performed using version SAS (Statistical Analysis) 7.2.7.2, with P < 0.05 as significant. The significance differences are expressed by letter notation: firstly, arranging all averages in sequence from large to small, and then, marking a letter a on the largest average; and comparing the average with a letter a until the average with a significant difference is marked, and marking a letter b; and then the average marked with b is used as a standard, and the letter b is marked uniformly even though the average marked with b is not obvious compared with the average above which is larger than the average marked with b; the maximum average marked with b is then used as a standard, and the letter b is continued to be marked without significance, compared with the unlabeled average below, until a certain average marked with significant difference c is encountered. The results are shown in Table 19, averaged for each group.
Table 19 animal feeding experiment effect comparison
| Group of | Initial weight/kg | Weight/kg | Daily weight gain/g | Ratio of material to weight | Diarrhea rate |
| Control group | 80.1 a | 91.4 a | 0.32 a | 6.5 a | 14.2 a |
| Experiment group one | 79.8 a | 94.1 b | 0.41 c | 4.5 b | 9.8 c |
| Experiment group II | 79.9 a | 93.8 b | 0.38 c | 5.2 b | 11.1 b |
Note that: the letters a, b and c on the same column of the digital shoulder marks represent that the difference is very significant (P < 0.01), and the same letter represents that the difference is not significant (P > 0.05).
As can be seen from the statistical analysis results in Table 19, the experimental group was significantly different from the control group in terms of average daily gain, weight-to-feed ratio, diarrhea rate (P < 0.05). The first experimental group has obvious difference with the second experimental group in reducing the calf diarrhea rate, and the addition amount is only half of that of the first experimental group. In addition, straw is used as a raw material in the solid state fermentation, and the cost of the solid state fermentation product is lower. Therefore, from comprehensive evaluation of feeding cost and growth performance, the product obtained by carrying out solid state fermentation on the straw by utilizing the supernatant of the combined fermentation of clostridium butyricum, lactobacillus acidophilus and saccharomyces cerevisiae can meet the requirements of part of calf breeding, and the effects of improving the growth performance of calves and reducing the diarrhea rate are achieved.
On the day of the end of the experiment, calves Niu Jinshi h, 5 calves jugular vein blood sampling is carried out for 5mL, centrifugation is carried out for 10min at 2500 r/min, upper serum is taken and stored at-20 ℃, and the content of immunoglobulin G (Ig G) in calf serum is measured.
The average value of the IgG content in the calf serum of the first experimental group is 9.96g/L, the average value of the IgG content in the calf serum of the second experimental group is 9.63g/L, the difference between the first experimental group and the second experimental group is obvious (P is less than 0.05), the average value of the IgG content in the calf serum of the control group is 7.84g/L, and the difference between the experimental group and the control group is obvious (P is less than 0.05).
From the results, it can be seen that the solid fermentation product obtained by using the mixed fermentation centrifugate can improve the growth performance of calves, improve the serum immunity level and reduce the diarrhea rate.
The last explanation is: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the 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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